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WO2025227129A2 - Delivery vehicles comprising proglucagon derived polypeptides and anabolic polypeptides and uses thereof - Google Patents

Delivery vehicles comprising proglucagon derived polypeptides and anabolic polypeptides and uses thereof

Info

Publication number
WO2025227129A2
WO2025227129A2 PCT/US2025/026532 US2025026532W WO2025227129A2 WO 2025227129 A2 WO2025227129 A2 WO 2025227129A2 US 2025026532 W US2025026532 W US 2025026532W WO 2025227129 A2 WO2025227129 A2 WO 2025227129A2
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
delivery vehicle
peptides
liposome
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/026532
Other languages
French (fr)
Other versions
WO2025227129A3 (en
Inventor
Jorge Luis Cabrera
Jorma A. Virtanen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Starrock Pharma LLC
Original Assignee
Starrock Pharma LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Starrock Pharma LLC filed Critical Starrock Pharma LLC
Publication of WO2025227129A2 publication Critical patent/WO2025227129A2/en
Publication of WO2025227129A3 publication Critical patent/WO2025227129A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
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    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
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    • A—HUMAN NECESSITIES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid

Definitions

  • Delivery vehicles comprising polypeptides for treating obesity are provided.
  • Therapies include administration combinatorial regimens, and administration of rotational combinatorial regimens.
  • the regimens comprise combinations of drugs and/or non-drug treatments.
  • Obesity is not confined to the United States. It is a global epidemic with serious medical and financial consequences.
  • the WHO formally recognized obesity as a global epidemic and viewed it as one of the most serious public health problems of the 21 st century.
  • the Organization for Economic and Co-operation Development has projected an increase in obesity rates world-wide until at least 2030, especially in the United States, Mexico and England with rates reaching 47%, 39% and 35%, respectively (“Obesity Update 2017,” published by the Organisation for Economic Co-operation and Development. Retrieved 6 October 2018).
  • the delivery vehicles display combinations of polypeptides for weight loss, such as proglucagon polypeptides, and a polypeptide that prevents or reduces muscle loss associated with weight loss or promotes muscle growth (referred to as a muscle enhancing polypeptide).
  • the delivery vehicles can contain or display other drugs, such as small molecule drugs, and they can contain the peptide drugs.
  • the vehicles are for delivery of combinations of drugs, including small molecules and peptides.
  • delivery vehicles where the delivery vehicles each display a polypeptide and/or contain a small molecule drug, and a plurality of delivery vehicles are co-formulated or administered together so that the combination of at least two weight loss promoting peptides and a muscle enhancing and optionally a small molecule drug, such as an appetite inhibiting drug, are administered together.
  • Devices for administering the delivery vehicles such as syringes or pens, that contain single or multiple dosage amounts of the delivery vehicles also provided.
  • the devices can contain multiple chambers, one containing a lyophilized composition containing the delivery vehicles or mixtures thereof, and a second containing a vehicle, such as PBS, for mixing with the delivery vehicles, for administration.
  • polypeptides linked to or embedded in the delivery vehicles can provided so that the combinations of polypeptides can be rotated, such as in regimens described in copending International PCT application No. PCT/US23/77508 (International PCT publication No. WO2024/091863).
  • delivery vehicles for effecting treatment of obesity and/or diabetes and for implementing the regimens and methods described in the copending application.
  • the polypeptides described therein can be linked to or embedded in delivery vehicles, such as liposomes or extracellular vesicles or vehicles, such as exosomes, for administration.
  • Two or three different polypeptides can be linked to or embedded in the delivery vehicle and administered, such as by injection, such as subcutaneous injection, and, depending upon the selected delivery vehicle by inhalation into the lungs or nose or by mucosal delivery, including orally.
  • Most liposomal preparations and exosomal preparations cannot be formulated for oral administration, but there are some that can be so-administered.
  • the delivery vehicles or combinations for delivery vehicles contain or display a polypeptide for weight loss and a polypeptide for muscle enhancement (prevention or reduction of muscle loss associated with weight loss and/or increasing muscle mass).
  • the delivery vehicles and composition include uses for treating obesity and/or diabetes, and/or other comorbidities, such as high cholesterol, heart disease, and hypertension, and other diseases, disorders, and conditions associated with metabolic syndrome.
  • All the methods and regimens and uses can provide the drug, generally a peptide drug, displayed on a delivery vehicle, such as a liposome or exosome.
  • the peptides can be linked, by standard well-known methods, to the delivery vehicle or the delivery vehicle can be produced with the peptide incorporated into the surface.
  • the delivery vehicles as detailed herein can display a plurality of peptides, or can display one or more and mixtures thereof can be administered or used together to provide a desired combination of peptides.
  • the methods, uses, and regimens herein are for treating obesity and/or diabetes.
  • the methods, regimens, and uses comprise a mixture of peptides for weight loss and for muscle enhancement for promoting or sustaining muscle growth, such as an anabolic peptide).
  • delivery vehicles comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle; the surface of the delivery optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and/or have different activities; and the therapeutic peptides target pathways involved in obesity and/or diabetes, or have activity for treating obesity and/or diabetes.
  • compositions comprising a mixture of delivery vehicles, where: each delivery displays at least one therapeutic peptide on the surface; and the composition comprises delivery vehicles selected so that the composition comprises at least three different displayed peptides.
  • composition can be formulated for any suitable route of administration, including, for example, intramuscular, intravenous, mucosal, parenteral, subcutaneous administration, oral, intranasal, inhalation, and other route.
  • the delivery vehicles and compositions are provided for subcutaneous administration. They can be provided as liquids or powders for reconstitution as a liquid.
  • Delivery vehicles include, but are not limited to liposomes, lipid nanoparticles, exosomes, and extracellular vesicles.
  • the peptides displayed in or on the delivery vehicles comprise fat loss and muscle enhancement peptides; as noted muscle enhancement includes muscle loss reduction or prevention and/or increased muscle mass, such as an anabolic peptide.
  • Exemplary of muscle enhancing peptides are sermorelin, tesamorelin, and IGF-1.
  • the muscle enhancement polypeptide by enhancing muscle growth prevents or reduces the loss of muscle that is a problem with administration of GLP-1 agonists.
  • at least two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide.
  • Polypeptides for fat loss can be selected from among: GLP-1, adiponectin, leptin, oxyntomodulin, PYY (peptide YY), amylin, pancreatic peptide, enterostatin/GIP (Gastroinhibitory Polypeptide), glicentin, glucagon, GRPP (glicentin-related pancreatic polypeptide), HGH (human growth hormone), CCK (cholecystokinin), neurotensin, secretin, IIP (myo-inositol 1 -phosphate), and MPGF (major proglucagon fragment).
  • GLP-1 adiponectin
  • leptin oxyntomodulin
  • PYY peptide YY
  • amylin pancreatic peptide
  • enterostatin/GIP Gastroinhibitory Polypeptide
  • glicentin glicentin
  • glucagon glucagon
  • GRPP glicentin-related pancre
  • Peptides for muscle enhancement include, but are not limited to, sermorelin, tesamorelin; and IGF1 (or HGH).
  • the combinations of drugs comprise peptides that comprise GLP-1, Oxyntomodulin, enterostatin/GIP (Gastroinhibitory Peptide); and Sermorelin.
  • the liposomes and other delivery vehicles also can contain or display small molecule weight loss drugs, such as phentermine.
  • Delivery vehicles include liposomes, such as large multilamellar vesicles (LMV) and SMVs (small MVs).
  • Liposomes include those that comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA).
  • Phospholipids include synthetic and phospholipids from natural sources, such as egg yolks. Cholesterol can be added to the liposomes to improve properties, such as permeability.
  • the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less.
  • the liposome can include lipids modified with a reactive group for coupling with a peptide or with a peptide modified with a reactive group.
  • the reactive group for the coupling reaction is selected from among amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and/or alkyne reactive groups.
  • the reactive group is an amino group that forms an amide bond with an activated carboxylic ester, or is a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or is a hydrazide that bind with carbonyl groups, or is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprise azide and alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the peptide or protein the liposome.
  • the delivery vehicle is a liposome and the peptide and/or liposome is/are PEGylated for linking the peptide to the liposome.
  • the peptide and/or liposome can be PEGylated for linkage, or linked to or coated with streptavidin for reaction with biotin, such as biotin linked to the peptide.
  • delivery vehicles and compositions where the peptide and liposome are linked via an amide/peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2,3-triazole linker.
  • the linkage between the delivery vehicle, such as liposome can comprise a spacer, such as, but are not limited to, a spacer that comprises polyethylene glycol (PEG) and/or and an oligonucleotides bound to the liposome and to the peptide linked to a complementary oligonucleotide.
  • the delivery vehicle, such as a liposome can comprise streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide.
  • the delivery vehicles such as liposomes
  • the delivery vehicles are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form liposomes that display the peptides upon mixing these peptide derivatives with streptavidin liposomes.
  • the linkage can include a spacer, such as a PEG spacer or oligonucleotide; one end of the spacer can be attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.
  • Containers comprising the delivery vehicles or compositions provided herein.
  • Containers include, for example, pens and syringes for administering the delivery vehicle.
  • compositions comprising the delivery vehicles provided herein.
  • the methods comprise administering a delivery vehicle or composition provided herein.
  • the methods can include the rotational combinatorial and rotational methods described herein, where the peptides are provided displayed on delivery vehicles, such as liposomes.
  • the disease, disorder, or condition contemplated for treatment is/are obesity and/or diabetes.
  • One combination of peptides can be administered, where the combination is displayed on the delivery vehicle.
  • a plurality of combinations can be administered where the different combinations are provided on delivery vehicles.
  • Mixtures of delivery vehicles displaying different peptides can be combined and administered.
  • Corresponding regimens, as described herein are provided. Provided are methods and regiments and delivery vehicles that comprise combinations of peptides mimic effects of gastric bypass.
  • the methods and regimens include a combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin.
  • GLP-1 glucagon-like peptide-1
  • PYY peptide YY
  • GIP enterostatin/gastric inhibitory peptide
  • the methods and regimens can further comprise a delivery vehicle that comprises a peptide that results in muscle enhancement, such as, for example, one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone.
  • muscle enhancement the loss of muscle associated with weight loss treatment that accompanies weight loss is reduced or prevented.
  • treated subjects, whether or not they exercise, have little or no muscle loss.
  • the methods and regimens can comprise administration of a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).
  • VIP vasoactive intestinal peptide
  • Peptides in the methods and regimens and linked to delivery vehicles can be selected from among: GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosinetyrosine (PYY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and/or the peptides are a GLP-1, Adiponectin, lept
  • the delivery vehicles also can contain and/or display a small molecule weight loss drug, such as an amphetamine or appetite suppressant, such as phentermine.
  • a small molecule weight loss drug such as an amphetamine or appetite suppressant, such as phentermine.
  • peptides that are displayed and small molecules that are incorporated into delivery vehicles are those selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alpha-glucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP-4) inhibitor), canagliflozin (sodium-glucose cotransporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist
  • the peptides can be selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.
  • the methods and regimens and/or delivery vehicles further can comprise a mitochondrial uncoupler linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle.
  • mitochondrial uncoupler include, for example, uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine).
  • UCP1 uncoupling protein 1
  • catecholamine a small molecule uncoupler
  • BAM15 2,4-dinitrophenol
  • BAM15 N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine.
  • the small molecule drugs which are available in oral dosage forms, such as phen
  • the methods and regimens herein include administering a plurality of different combinations of drugs, wherein: each combination of drugs is administered for a predetermined time; each combination of drugs is rotated until all combinations are administered at least once to comprise a cycle; a cycle comprises at least two different combinations; a combination comprises at least two different drugs that target different pathways or targets involved in the disease, disorder, or condition; each combination of drugs is unique among the combinations administered in a cycle, but a drug can be part of a plurality of combinations as long as the resulting combinations are unique; the disease, disorder, or condition is a chronic disease, disorder, or condition that requires treatment for at least 6 months; and the disease, disorder, or condition is not a cancer.
  • Delivery vehicles containing/displaying a combinations of the peptides, and optionally a small molecule(s), such as phentermine or a drug to treat a comorbidity of obesity, such as metabolic syndrome, diabetes, high cholesterol, heart disease, and hypertension are provided.
  • a delivery vehicle such as a liposome or an exosome
  • examples of combinations of polypeptides for displaying on or delivering in a delivery vehicle, such as a liposome or an exosome include combinations of at least two or three weight loss drugs and a muscle enhancer, and can further include a small drug, such as an appetite suppressor, an amphetamine family drug, and/or drugs for treating obesity co-morbidities.
  • the small molecules can be fabricated in association with the liposomes, or can be separately administered. Examples of fabrication of liposomes is detailed in the above examples, and known and/or apparent to those skill in the art. Exemplary combinations of peptides and sequences are as follow:
  • Drug 1 GLPl/GIPl/Oxyntomodulin + ME* (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44
  • Drug 2 GLPl/GIPl/Amylin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44 Drug 3: GLP1/GIP1 /Glucagon + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44 Drug 4: GLP1/GIP1/CCK+ ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44 Drug 5: GLP1/GIP1/PYY + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 6 or
  • Different delivery vehicles can be rotated to provide rotational combinatorial treatment.
  • rotational combinatorial regimens for treating a disease, disorder, or condition comprising a plurality of combinations of drugs and/or treatments for a disease, disorder, or condition, wherein: the disease, disorder, or condition has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the rotational combinatorial therapy comprises at least two different combinations of drugs and/or treatment; the drugs and/or treatments in each combination target different pathways or targets involved in the disease, disorder, or condition; each combination is administered at least once a cycle; each cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; the cycle for each combination can be the same or a different length of time; and at least one of the combinations comprises at least two different drugs that target different targets or pathways.
  • a disease, disorder, or condition comprising administering a drug regimen comprising serially administering two or more combinations of drugs and/or treatments, by administering the delivery vehicles provided herein, wherein: the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the drug regimen comprises at least two combinations; at least one combination in the regimen includes at least two drugs that treat at least one target or pathway involved in the disease, disorder, or condition; the at least two drugs treat different targets, pathways, and/or have different activities.
  • combinatorial therapeutic methods of treating a disease, disorder, or condition comprising administering a combination of at least 2 or 3 different drugs selected from among a plurality of drugs, and generally including a muscle enhancer; wherein the disease, disorder, or condition is a chronic disease, disorder, or condition; the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; each of the plurality of drugs can treat a target or pathway involved in the disease, disorder, or condition; each of the selected drugs has a different activity from the other drugs in the combination; and the combination reduces or eliminates desensitization to one or more of the selected drugs.
  • the methods and regimens are those where the drugs and/or treatments in each combination are administered simultaneously, sequentially, or intermittently.
  • the drugs/treatments in a combination they can be administered together, simultaneously, serially, or intermittently. They can be administered within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or longer, such as up to 2 or 3 days.
  • the drugs and treatments in each combination are administered within 24 hours.
  • the methods and regimens include those where the disease, disorder, or condition is selected from a disease, disorder, or condition that includes treatments that when used as a monotherapy, the subject becomes desensitized to the drug.
  • the rotational methods herein can avoid desensitization.
  • the methods and regimens can comprise at least 2, 3, or more cycles, whereby treatment lasts at least 6 months, 9 months, a year, or longer.
  • rotational combinatorial methods and regimens that comprise at least 3 different combinations in a cycle. Included are methods and regimens where each combination of drugs and/or treatments comprises at least 2 different drugs or treatments that target different pathways or targets for intervention. In some embodiments, the drugs and treatments in a combination or in more than one combination can target the same pathway or target for intervention.
  • the cancer is not among the disease, disorder, or condition that is treated for which the regimen is employed.
  • a delivery vehicle comprising a muscle enhancing polypeptide, is administered to prevent or reduce loss of muscle associated with muscle or to enhance muscle growth.
  • Regimens for treating obesity comprising a combination or combinations of drugs whose effects or activities mimic the biological effects of gastric bypass surgery, such as methods and regimens where biological effects of gastric bypass comprise reduced absorption and/or malabsorption of food, decreased appetite, increased satiety, increased glycogenolysis and/or lipolysis, increased insulin sensitivity, modulation of energy expenditure, and inhibition of gastric emptying.
  • Such methods and regimens can comprise or further comprise drugs that increase growth hormone, and/or promote or result in muscle enhancement so that at least the loss of muscle associated with weight loss is reduced or eliminated.
  • each combination can be administered for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least one month, or at least two months, or at least 3 months.
  • Each combination can be administered for the same predetermined length of time, or each combination can be administered for a different length of time, or at least one of the combinations can be administered for a different length of time from the other combinations.
  • drug regimens for use for treating obesity comprising a combination or combinations of drugs or drugs and treatments whose combined effects mimic gastric bypass, and optionally comprising additional drugs that promote or result in weight loss, wherein each combination comprises at least three different drugs that target a different pathway or intervention target involved in the etiology of obesity.
  • the methods and regimens and uses can comprise one or more of a drug that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide- 1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and Glicentin; a drug that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; a drug that modulates energy expenditure comprising a drug selected from among leptin, oxyntomodulin, and glicentin; a drug that results in muscle enhancement comprising one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone; and a drug that promotes intestinal smooth muscle relaxation comprising vasoactive intestinal peptide (VIP).
  • the combinatorial methods, regimens and uses can be those where the drugs are selected from among drugs that have activities or effects selected from among: drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating or appetite, inhibit or modulate gastric acid secretion, limit or decrease the rate of gastric emptying, enhance muscles, increase glycogenolysis, increase insulin sensitivity, enhance the body weightlowering and/or glucose-lowering efficacy of GLP-1, medication(s) or therapy that decrease ghrelin or ghrelin-associated activation pathways, and drugs and treatments that reduce or antagonize ghrelin.
  • Exemplary combinations of drugs include combinations of drugs that are selected from among GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin/glucose-dependent insulinotropic peptide or gastric inhibitory polypeptide (GIP,) cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15).
  • exemplary combinatorial methods, regimens, uses include those where the drugs that are combined comprise a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP-4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, drugs that increase gastric inhibitory polypeptide (GIP2), and mitochondrial uncouplers, such as, but not limited to those in which the drugs are selected from among one or more of dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase
  • the methods, regimens, and uses include those comprising a mitochondrial uncoupler, such as, for example, where the mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM 15 ((2 -fluorophenyl) ⁇ 6- [(2-fluorophenyl)amino]( 1 ,2,5 -oxadi azolo [3 ,4e] pyrazin-5-yl) ⁇ amine).
  • UCP1 uncoupling protein 1
  • DNP 2,4-dinitrophenol
  • BAM 15 BAM 15
  • the small molecule drugs which are available in oral dosage forms, such as phentermine, can be co-administered with the liposomes containing or displaying the polypeptides.
  • the combination or each combination of drugs comprises at least three drugs that target different pathways or targets for intervention. These include those in which the disease, disorder, or condition requires treatment for at least 6 months, 9 months, or 1 year, or longer, or longer and indefinitely, or for life.
  • kits for preventing (or reducing the risk) of desensitization to treatments for a disease, disorder, or condition comprising administering a rotational combinatorial therapeutic regimen.
  • Such methods employ the methods and regimens and uses described above and elsewhere herein.
  • the combinations of drugs and treatments can be administered together, sequentially, intermittently, and/or within a predetermined time period, such as within up to a 24- hour period, or up to a 12-hour period, or up to a 6-hour period or less.
  • Exemplary of the methods, regimens and uses is a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance muscles, and inhibit gastric emptying; b) a second combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and promote muscle enhancement.
  • a first combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance muscles, and inhibit gastric emptying
  • a second combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis
  • a third combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance the body weight-lowering and glucose- lowering efficacy of GLP-1, and promote muscle enhancement.
  • any of the methods, regimens, and uses herein can be a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and enhance or promote muscle.
  • a method, use, or rotational combinatorial regimen for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination that comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination that comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-and lowering efficacy of GLP- 1, and enhance or promote muscles.
  • the regimen, method, or use comprises the following combinations or administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement.
  • the rotational combinatorial regimen, method, or use for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement.
  • the method or regimen or use can comprise combinations in each combination or in each cycle administered for 2-, 3-, 4-, 5-, or 6-month intervals.
  • the intervals between or for each administration of each combination are the same or vary.
  • at least one combination in a cycle can comprise at least three different drugs.
  • at least one combination in a cycle comprises at least four different drugs; and optionally at least one combination includes only a single drug.
  • delivery vehicles including liposomes and exosomes, compositions, regimens, methods, uses, combinations, kits, and articles of manufacture as follows and/or including variations thereof within the skill in the art.
  • delivery vehicles comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle or in the delivery vehicle; the surface of the delivery vehicle optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and/or have different activities; and the peptides target pathways involved in obesity and/or diabetes, or have activity for treating obesity and/or diabetes or other obesity comorbidity.
  • These vehicles can further comprise a small molecule drug for treatment of obesity or an associated comorbidity.
  • compositions comprising a mixture of delivery vehicles, where: each delivery vehicle displays at least one of the weight loss or fat loss peptides on the surface and/or contains the at least one peptide; and the composition comprises delivery vehicles that are selected so that the composition comprises at least three different peptides.
  • the compositions can be formulated for a suitable route of administration, including injection, oral, and intravenous.
  • the delivery vehicles are administered by subcutaneous (SC) injection in a volume of less than about 5 mL or 3 mL, or in a volume of 10 mL or more, particularly if formulated with an excipient the facilitates administration, such as a hyaluronidase.
  • SC subcutaneous
  • Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles (LPNs), exosomes, and other extracellular vesicles.
  • LPNs lipid nanoparticles
  • exosomes and other extracellular vesicles.
  • the peptides in or on the delivery vehicles or in compositions or for combination therapy or for administration with the delivery vehicles comprise fat loss and muscle enhancement peptides, wherein muscle enhancement polypeptides reduce or eliminate loss of muscle mass or increase muscle mass.
  • Additional drugs for administration include small molecule drugs, which can be incorporated in or on or associated with the delivery vehicle (such as by non-covalent interaction), for weight loss, for treatment of comorbidities, such as hypertension, high cholesterol, and diabetes, associated with obesity, and or for treating other diseases, disorders, and conditions.
  • Exemplary of the delivery vehicles are those where two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide.
  • PeptideFL 2 Adiponectin
  • PeptideFL3 Leptin
  • PeptideFL4 Oxyntomodulin
  • PeptideFL5 PYY
  • PeptideFL6 Amylin
  • PeptideFL7 Pancreatic peptide
  • PeptideFL8 Enterostatin/GIP (Gastroinhibitory Polypeptide)
  • PeptideFL9 Glicentin
  • PeptideFL 10 Glucagon
  • PeptideFL 11 GRPP
  • PeptideFL 12 HGH 176-191
  • Peptide FL13 CCK
  • PeptideFL 14 Neurotensin
  • PeptideFL15 Secretin
  • PeptideFL 16 IP1
  • PeptideFL17 MPGF (major proglucagon fragment); and optionally a muscle enhancing polypeptide and/or a drug such as a myostatin pathway inhibitor, such as an antibody, such as bimagrumab, that inhibits muscle wasting.
  • PeptideME2 Tesamorelin
  • PeptideME2 IGFlor human growth hormone to induce IGF1.
  • a myostatin pathway inhibitor can be included, such as for example, an antibody or an antigenbinding fragment thereof that competes with apitegromab for antigen binding.
  • myostatin pathway inhibitors include, but are not limited to, a ligand trap (e.g., ACE- 031, ACE-083, and BIIB-110/ALG-801); an anti-ActRIIb antibody (e.g., bimagrumab); a neutralizing anti-myostatin antibody (e.g., stamulumab (MYO-029), domagrozumab (PF-06252616), or Landogrozumab (LY2495655)), a myostatin peptibody (e.g., AMG-745/PINTA-745), or an anti -myostatin adnectin (e.g., RG6206 or BMS-986089 (also known as taldefgrobep alfa)); wherein, further optionally, the non-selective myostatin pathway inhibitor also inhibits Activin A and/or GDF11 (see, US20240368262, which describes myostatin pathway inhibitor
  • the peptides for delivery and/or administration are peptides for fat loss (FL) and optionally peptides for ME, where: a) the polypeptides for fat loss are selected from among:
  • PeptideFL 1 GLP-1
  • PeptideFL 2 Adiponectin
  • PeptideFL3 Leptin
  • PeptideFL4 Oxyntomodulin
  • PeptideFL5 PYY
  • PeptideFL6 Amylin
  • PeptideFL7 Pancreatic peptide
  • PeptideFL8 Enterostatin/GIP (Gastroinhibitory Polypeptide),
  • PeptideFL9 Glicentin
  • PeptideFL 10 Glucagon
  • PeptideFL 12 HGH 176-191
  • PeptideFL 14 Neurotensin
  • PeptideFL15 Secretin
  • PeptideFL 17 MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement are selected from among:
  • PeptideMEl Sermorelin
  • PeptideME2 Tesamorelin
  • PeptideME2 IGFl (or human growth hormone to induce IGF1).
  • Exemplary combinations include those, for example, where the peptides comprise GLP-1, Oxyntomodulin, enterostatin/GIP (Gastroinhibitory Peptide); and Sermorelin, and other combinations as detailed herein.
  • the peptides and other drugs can be exogenously introduced into and/or onto the vehicle or fabricated with the vehicle so that the peptide/drug is associated with or in the vehicle by any suitable interaction, including covalent bonding and non-covalent interactions.
  • Delivery vehicles include, but are not limited to, liposomes, including liposomes that are large multilamellar vesicles (LMVs).
  • the liposome can comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA).
  • phospholipids such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA).
  • PC phosphatidyl choline
  • PE phosphatidyl ethanol amine
  • PS phosphatidyl serine
  • PA phosphatidic acid
  • Liposomes can be from the phospholipid is from a natural source and loaded with the peptides and drugs or reacted to become associated with the peptide
  • the liposomes can cholesterol, particularly so that the amount of cholesterol is sufficient to increase the permeability of the liposome compared to the liposome that does not contain the cholesterol, such as for example, where the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less.
  • the liposome or other vehicle can lipids or other molecules modified with a reactive group for coupling with a peptide or peptide modified with a reactive group, such as for coupling with a peptide or peptide modified with a reactive group.
  • Exemplar reactive groups for the coupling reaction include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne groups.
  • the liposome can comprise lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group.
  • Reactive groups include, but are not limited to, an amino group that forms an amide bond with an activated carboxylic ester, or a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or a hydrazide that binds with carbonyl groups, or a is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprises azide and/or alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the protein to the liposome.
  • Other delivery vehicles and peptides can be similarly modified as appropriate.
  • the peptides can be linked to the vehicle via the bonds formed by reaction of the reactive groups.
  • liposomes or other vehicle where the peptide and liposome (or other vehicle) are linked via an amide/peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2, 3 -triazole linker.
  • the peptide and/or vehicle, such as the liposome is/are PEGylated for linking the peptide to the liposome.
  • the linkage of the peptide to the delivery vehicle, such as the liposome comprises a spacer, such as, but are not limited to, polyethylene glycol (PEG) and/or and an oligonucleotides bound to the vehicle, such as a liposome, and to the peptide linked to a complementary oligonucleotide.
  • vehicle such as the liposome, comprises streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide.
  • the vehicle such as the liposomes
  • the vehicle are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form vehicles, such as liposomes, that display the peptides upon mixing these peptide derivatives with streptavidin liposomes.
  • the linkage can comprise a PEG spacer, where one end of the spacer is attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.
  • the delivery vehicle or compositions provided herein that comprises one or more small molecule drug(s), where: the small molecule drug enhances weight loss or treats a comorbidity associated with obesity; and the small molecule is formulated in or with the delivery vehicle or is for administrations separately from the delivery vehicle.
  • Exemplary small molecule drug(s) is/are selected from among one or more of: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate.
  • the delivery vehicle and small molecule drugs can be for administration together in a single composition or for administration in separate compositions for administration at substantially the same time, sequentially, or intermittently, or in accord with a particular combination therapy regimen, in which the drugs are administered in a particular order and/or according to a particular schedule.
  • Regimens for administration of the combinations of the drugs/peptides are provided.
  • containers that comprise the delivery vehicle or compositions or combinations provided herein.
  • exemplary of containers are vials, pens, and syringes, including injectors, such as autoinjectors.
  • Autoinjectors are known that provide for self-administration. Included are autoinjectors are multicompartment containers, where one compartment contains the delivery vehicles or a mixture of different delivery vehicles. They can be provided in a pharmaceutically acceptable vehicle or can be lyophilized.
  • the peptides/drugs are provided as lyophilized powders, and there is a separate compartment that contains the pharmaceutically accept vehicle for solubilizing or dissolving the powders to produce a solution or suspension, such as microemulsion, suitable for injection, particularly subcutaneous injection.
  • a pharmaceutical compositions containing any of the delivery vehicles and mixtures there in a pharmaceutically acceptable vehicle for administration are provided. These compositions for administration or for use in methods for treating obesity and/or associated diseases, disorders, and conditions. Hence, methods for treating obesity as well as other comorbidities and uses for the vehicles, compositions, and combinations are provided. For example, provided are methods and uses for treatment of obesity or diabetes in which a delivery vehicle, and/or composition and/or combination provided herein is/are administered. This includes combinatorial and rotational methods, such as those, where the drugs include peptides and optionally small molecule drugs; and the disease, disorder, or condition is obesity and/or diabetes; where at least two combinations are rotated for each cycle of treatment; and treatment comprises at least two cycles.
  • Regiments for treating a disease, disorder, or condition comprising combinations of peptides for use in a rotational combinatorial regimen are provided, such as where the disease, disorder, or condition is obesity or diabetes, or other obesity comorbidity, or other chronic disease, disorder, or condition that requires treatment for at least 6 months; each combination comprises at least two different peptides that target different pathways or intervention targets; the combinations of peptides comprise a delivery vehicle or composition provided herein.
  • the regimen can include a small molecule drug or drugs for weight loss and/or for treating a comorbidity associated with obesity or other chronic condition.
  • the small molecule drug can be selected from one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
  • the small molecule drug can be incorporated in or on the delivery vehicle and/or the small molecule drug is for co-administration, simultaneously, sequentially, or intermittently with the delivery vehicle(s).
  • the methods can be a rotational combinatorial method in which combinations of peptides and/or drugs are rotated.
  • Such methods and regimens can comprise a plurality of cycles of administration of different combinations of peptides and optionally the small molecules, where each combination is administered at least once a cycle; a cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; and at least one of the combinations comprises at least two different drugs that target different targets for intervention or pathways.
  • each cycle can comprise one delivery vehicle that comprises at least 3 different peptides and optionally small molecule drugs, or mixtures of delivery vehicles that comprise different peptides and/or small molecule drugs, each delivery vehicle comprises at least peptide, whereby at least three different peptides are administered in each cycle; and the different delivery vehicles are administered together or serially.
  • the small molecule drugs include, for example, one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
  • Different vehicles or combinations of vehicles and/or drugs can be administered within 24 hours or other suitable time period determined by the skilled person, of each other.
  • each peptide can be selected to targets a different pathway or different target for intervention for treatment of obesity.
  • the combinations of peptides mimic all or some of the effects of gastric bypass.
  • Exemplary combinations of peptides include, but are not limited to, combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates
  • the vehicle can comprise and/or display and/or contain combinations of peptides selected from among combinations of drugs for fat loss + a drug for muscle enhancement as follows: a) GLP1 /GIP 1 /Oxyntomodulin + Sermorelin or Tesamorelin or IGF1; b) GLPl/GIPl/Amylin + Sermorelin or Tesamorelin or IGF1; c) GLP1 /GIP 1 /Glucagon + Sermorelin or Tesamorelin or IGF1; d) GLP1/GIP1/CCK+ Sermorelin or Tesamorelin or IGF1; e) GLP1/GIP1/PYY + Sermorelin or Tesamorelin or IGF1; and f) GLP1 /GIP 1 /Leptin + Sermorelin or Tesamorelin or IGF1.
  • Exemplary amino acid sequences of each of the peptides for each of a)-f) are as set forth set forth in the following SEQ IDs, or are portions, or are variants thereof that have at least 90% or at least 95% sequence identity thereto and retain activity for effecting fat loss or muscle enhancement: a) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44; b) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44; c) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44; d) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44; e) SEQ ID NOs: 1, 10, 6 or 36 + SEQ ID NOs: 5, 8, 44; and f) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44.
  • the delivery vehicle, composition, combination, method, and/or regimen includes a peptide that results in muscle enhancement or can include a peptide or other molecule that inhibits or reduces muscle loss, such as a myostatin pathway inhibitor.
  • exemplary muscle enhancement peptides and drugs include, but are not limited to, one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone.
  • the herein the delivery vehicle, composition, combination, method, and/or regimen can comprise a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).
  • VIP vasoactive intestinal peptide
  • Peptides can be selected from among: a) GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (EC A), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and/or b) the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone,
  • Exemplary of embodiments herein are those in which the peptides and drugs are selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin (SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin.
  • dulaglutide bydureon
  • semaglutide semaglutide
  • exenatide liraglutide
  • phentermine liothyronine
  • topiramate carbonic anhydrase inhibitor
  • the peptides and drugs are selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.
  • the delivery vehicle, composition, method, use, or regimen can include or further comprise a mitochondrial uncoupler, wherein the mitochondrial uncoupler is provided linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle, such as a mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2- Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine).
  • UCP1 uncoupling protein 1
  • DNP 2,4-dinitrophenol
  • BAM15 2,4-dinitrophenol
  • BAM15 N5,N6-bis(2- Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine
  • a PEGylated peptide comprising preparing a PEGylated lysine and adding it to peptide during solid phase synthesis, where optionally the PEGylated lysine is prepared by reacting the epsilon-amino group of the lysine with a carboxyl terminated PEG moiety via an amide group to produce a PEGylated lysine.
  • the peptides can be modified by replacing lysines that are not intended to be PEGylated with a conservative amino acid.
  • the methods of synthesis can include adding the PEGylated lysine to a peptide during solid phase synthesis of the peptide.
  • the PEG moiety comprises a functional group for conjugation with a reactive group, such as one on a liposome. Functional groups include, but are not limited to, acetylene that is reacted with azide in the liposome (click chemistry).
  • Other methods of conjugating a polypeptide to a liposome or other delivery vehicle comprise: adding a lysine to a polypeptide during solid phase synthesis by reacting the epsilon-amino group of lysine with a carboxyl terminated PEG moiety via an amide group to produce a peptide comprising a lysine comprising the PEG moiety, which comprises a functional group for conjugation with the liposome; and conjugating the PEGylated peptide to the liposome.
  • the functional group such as acetylene, can be reacted, for example, with azide fabricated in or added to the delivery vehicle, such a liposome, thereby employing click chemistry.
  • any of the delivery vehicles provided herein can be so-prepared, including where the peptide is a GLP-1 pathway agonist and/or a muscle enhancer peptide, or any other combinations as described herein or that are apparent to the skilled artisan from the description herein.
  • the method can include prepared at least three pegylated peptides and linking the peptides to one delivery vehicle, such as a liposome, or each to a different delivery vehicle, such as a liposome, or two can be lined to one delivery vehicle, such as a liposome, and the third on a separate delivery vehicle, such as a liposome, or other combinations, or all can be linked or introduced into or onto a single delivery vehicle, such as a liposome.
  • peptides linked to the delivery vehicles such as a liposome, or to different delivery vehicles, such as liposomes, wherein a least two of the peptides are for fat loss, and one is for muscle enhancement.
  • peptides can be selected where: a) the peptides for fat loss (FL) are selected from among:
  • PeptideFL 1 GLP-1
  • PeptideFL 2 Adiponectin
  • PeptideFL Leptin
  • PeptideFL4 Oxyntomodulin
  • PeptideFL5 PYY
  • PeptideFL6 Amylin
  • PeptideFL7 Pancreatic peptide
  • PeptideFL8 Enterostatin/GIP (Gastroinhibitory Polypeptide),
  • PeptideFL9 Glicentin
  • PeptideFL 14 Neurotensin
  • PeptideFL15 Secretin
  • PeptideME2 Tesamorelin
  • PeptideME2 IGFl (or human growth hormone to induce IGF1).
  • delivery vehicles such as liposomes that comprise one or more of a fat loss peptide and a muscle enhancement peptide produced by any of the above methods or methods that in light of the disclosure herein are known to the skilled artisan.
  • the delivery vehicle, composition, use, method, regimen, or liposome can include a small molecule drug, such as where the drug is for weight loss and/or a co-morbidity associated with obesity.
  • Comorbidities include, but are not limited to, diabetes, hypertension, high cholesterol and/or other elevated lipids (dyslipidemia), metabolic syndrome, and heart disease.
  • Small molecule drugs include, but are not limited to, Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
  • the products and methods can include phentermine, including regiments and methods where the phentermine is provided in or on the delivery vehicle, or wherein phentermine is provided as a separate composition, such as in a tablet, capsule, gel, or other form for oral administration, for combination therapy.
  • the delivery vehicles, compositions, combinations, uses, method, regimen the delivery vehicle can be an extracellular vesicle or an LPN, or an exosome, or a liposome.
  • kits and articles of manufacture containing the delivery vehicles and containers and compositions and combinations.
  • Figure 1 depicts bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1/3 and PC2.
  • GLP-1 and GLP-2 glucagon-like peptides -1 and -2
  • oxyntomodulin es glicentin and glicentin-related pancreatic peptide
  • PC prohormone convertase
  • Figures 2A and 2B depict a liposome with linkages and linked moieties.
  • Figure 3 depicts PEG (X) with an N-hydroxy succinimide active ester on one end.
  • Figure 4 depicts an exemplary coupling chemistry in which X is Br or I.
  • Figures 5A-C depict a polypeptide with free lysines and Pegylation thereof.
  • Figure 5A depicts a short segment of a polypeptide, such as any of the GLP-1 agonist polypeptides and/or a muscle enhancement polypeptide, which is Pegylated as shown herein
  • Figure 5B depicts an exemplary resulting product in which a lysine is PEGylated.
  • the PEG linked to the lysine has an azide group at the end for coupling to a liposome that has an acetylene group on the surface.
  • Figure 5C depicts the product.
  • PGDP Proglucagon derived Peptides
  • muscle enhancement peptides a.
  • Proglucagon Derived Peptides and tissue specific secretion b.
  • Unimolecular multi-agonists c. Enhancing fat loss efficacy via proglucagon derived peptides
  • Combination Therapies to Treat Overweight and Obesity 1. Therapeutic Uses of the Combinatorial Therapy a. Combination Therapies in Cancer b. Combination Therapies in Pain Management c. Combination Therapies in Oral Contraception d. Combination Therapies to Treat Pathogens e. Combination Therapies to Treat Alzheimer’s Disease f. Combination Therapies to Treat Hypertension g. Combination Therapies to Treat Parkinson’s Disease h. Combination Therapies to Treat Chronic Obstructive Pulmonary Disease (COPD) i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions j. Combination Therapies to Treat Overweight and Obesity
  • COPD Chronic Obstructive Pulmonary Disease
  • Amphetamines e.g., phentermine-topiramate
  • Lipase inhibitors e.g., Orlistat
  • Serotonergic agonists e.g., lorcaserin
  • Neuromodulators e.g., lorcaserin
  • Bupropion/Naltrexone such as the product sold under the trademark Contrave®
  • GLP1R Glucagon-like peptide-1 receptor
  • Mitochondrial uncouplers g. Thyroid hormones
  • Drug cocktails i. Cannabinoid receptor antagonists
  • a delivery vehicle refers to macro-molecular structures in an emulsion, such as micelles, inverse micelles, lipid bilayers (liposomes) and cubosomes or a mixture thereof, as well as lipid nanoparticles and other lipid-based vehicles.
  • Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles, extracellular vehicles, such as exosomes.
  • the polypeptides are displayed on the surface of the vehicles such as by linkage to moieties on the surface.
  • the polypeptides can be embedded in the surface when the delivery vehicle is formed; and/or can be expresses in a membrane protein in vivo, such as during formation or preparation of an extracellular vehicle.
  • therapies used interchangeably with treatments include drugs and can include other non-drug treatments.
  • treatments include drugs (or medications) and non-drug treatments, such as, for example, surgery.
  • drugs include any administered therapeutic and is used interchangeably with medications.
  • a therapeutic agent is used interchangeably with therapeutic and includes, but is not limited to: conventional drugs and drug therapies; vaccines; pharmaceutical medications; pharmaceuticals; homeopathic medications; peptides; protein therapeutics; radio-protectants; radiation therapy; and chemotherapeutics.
  • a therapeutic agent can effect amelioration of symptoms of a disease, disorder, and/or condition or can prevent or reduce the risk of developing a disease, disorder, or condition, or reduce the severity of disease, disorder, or condition.
  • the combinations when used for the combinatorial therapy as described herein include at least 3 treatments, such as three different therapeutics.
  • the combinations can include 2 treatments in a combination or combinations of treatments. Some of the rotations can include a single drug or therapy, as long as one or more others rotational combinations include at least two. Generally, each rotated combination includes at least two different drugs or treatments.
  • combinatorial therapy or combinatorial protocol or regimen refers to combinations of treatments for a disease, disorder, or condition from among a plurality of treatments, at least 2 and generally at least 3, where each treatment acts on or interacts with or modulates a different target molecule and/or target pathway involved in or that mediates the disease, disorder, or condition.
  • a rotational combinatorial therapy is used interchangeably with combinatorial rotational treatment, or rotational combinatorial therapy, or rotational combination therapy, or rotational combination treatment or CRT, or grammatical variations thereof.
  • a rotational combinatorial therapy or protocol or regimen includes at least two different therapeutic combinations at least one of which, and generally each of which, includes at least two therapeutics or therapies known to activate different molecular and/or cellular pathways or targets involved in a disease, disorder, or condition.
  • Combinatorial rotational therapy thus, is a combinatorial therapy protocol that includes a plurality of rounds of treatment with different combinations of treatments or with the same combination but not successively. The different combinations are therapeutics are administered for a predetermined time, and then rotated for another predetermined time.
  • the number of rotations of combinations can be two, three, four, five or more, and can be administered for weeks, months, years, and indefinitely depending upon the disease, disorder, or condition.
  • the predetermined time can be days or weeks or months, generally is about 2-6 weeks. Each predetermined time is not necessarily the same.
  • a rotational combinatorial pharmacological treatment also referred to a rotational combinatorial therapy refers to a combinatorial therapy or treatment with pharmaceuticals or medications or drugs or treatments in the combinations where combination are rotated.
  • the rotational combinatorial pharmacological treatment includes a plurality of combinations of a plurality of pharmaceuticals or medications or drugs that are rotated in accord with a rotational regimen or protocol.
  • the rotational regimen generally is employed to prevent desensitization to a particular drug.
  • a rotational combinatorial regimen herein includes two or more different therapies or therapeutics (e.g., combinations of therapies and/or therapeutics) that are administered such that the combinations of the therapies are rotated for a plurality of rounds of treatment. Where there is a plurality of rounds of treatment, at least one round can include only a single treatment.
  • the rounds include combinations of at least two different treatments, where each targets a different pathway involved in the disease, disorder, or condition.
  • rotate or rotational or grammatical variations thereof include an exchange of one treatment for another.
  • a combinatorial rotation refers to changing one therapeutic combination to another, which has the effect of improving the therapeutic response, such as by avoiding desensitization to a therapeutic of combination thereof. This also can reduce adverse side effects, and/or the severity of adverse side effects.
  • a rotation includes a regimen in which a first combination of more than one therapeutics is administered, and the therapy is rotated (or switched) to a second combination one or more one therapeutic, and, optionally, the therapy is rotated (or switched) to a third combination of more than one therapeutics.
  • the rotations occur according to a predetermined time schedule, or in view of a physician’s judgement, such as based on an observation that a combination has reduced effect.
  • the therapies are rotated more than three times, such as more than four times, more than five times, more than six times, more than seven times, more than eight times, more than nine times, or more. In some instances, such as chronic diseases, disorders, and conditions, the rotation of therapies continues for up to life.
  • a regimen is used interchangeably with protocol and refers to a course of medical treatment. In some examples the regimen is designed to improve or preserve the health of the patient or to attain a particular result.
  • a therapeutic regimen or protocol includes the timing for administration of the therapeutics, timing for cessation of therapeutics, therapeutic dosage(s), the particular therapeutics that are included in particular combinations, and other components of experimental design, such as how therapeutics are administered.
  • a protocol can include provisions for how the protocol can be modified, such as, for example, in the case of receptor downregulation or an increase in adverse side effects.
  • a cycle is generally a series of events that are repeated regularly in the same order.
  • a cycle refers to the repeated administration of combinations of drugs, where a combination of drugs (or a drug) is administered for a period of time followed by another combination of drugs, and so on until all combinations in a cycle of treatment are administered.
  • the cycle then can be repeated; generally, the same combinations of drugs or treatments are administered. It, however, is within the discretion of the physician to change the drugs/treatments in accord response(s) of the treated subject, such as to improve therapeutic effectiveness, eliminate or reduce desensitization, and/or for other reasons, such as to reduce adverse side effects.
  • a combination can include a single drug, as long as at least one combination in the cycle includes two or more drugs or treatments.
  • disease or disorder or condition refers to a pathological or undesirable or undesired state in an organism resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and that is characterized by identifiable symptoms. Obesity and overweight are conditions characterized by excess body weight.
  • mucosal delivery refers to delivery of an agent in which the agent is introduced to the body across a mucous membrane which allows for the avoidance of the gastrointestinal tract and first pass liver metabolism and consequently allows the agent to directly enter circulation. This can include passage through the gastrointestinal tract as by oral ingestion, but refers to delivery through the mucosa of such locus.
  • a chronic condition is one that is expected, based on physician’s experience and knowledge in the art to last more than 6 months, and generally more than a year. It is a condition that requires treatment for at least 6 months, and can require at 1 year or more, including for life.
  • treating a subject with a disease, disorder, or condition means that a drug, therapeutic, composition, combination or other therapy is administered to the subject and the subject’s symptoms or manifestations of the disease or conditions are partially or totally ameliorated, or remain static (do not worsen) following treatment.
  • treating a subject with a disease, disorder, or condition includes treating a subject with a rotational combinatorial therapy described herein.
  • a pathway refers to a biological pathway that is involved in or mediates disease, disorder, or condition.
  • a pathway can be targeted via a component of the pathway, such as a receptor or ligand involved in the pathway.
  • the targets can be antagonized or agonized.
  • a “combination” refers to any association between two or among more items.
  • the association can be spatial or refer to the use of the two or more items for a common purpose.
  • a combination for therapeutic use(s), such as a rotational combinatorial therapy provided herein includes more than one therapeutic (z.e., medication), such as two, three, four, five, or more therapeutics, but generally includes up to five therapeutics in the combination.
  • the rotational combinatorial therapy provided herein includes a regimen in which two, three, four, five or more combinations are rotated.
  • the combinations for rotation in the regimen provided herein can contain different therapeutics or can contain some of the same therapeutics, but do not contain all of the same therapeutics in the combinations.
  • a “combination” is used herein interchangeably with a cluster, which includes two or more treatments, such as two or more medications.
  • Each combination can be administered as a therapy, or can be rotated and administered for a predetermined time, which can be shortened or lengthened according to the judgement of a physician. When not rotated each combination generally includes at least 3 different treatments
  • treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Hence, treatment encompasses prophylaxis, therapy and/or cure. Treatment also encompasses any pharmaceutical use of the compositions and combinations herein. Treatment also encompasses any pharmaceutical use of a combination of pharmaceutical and non-pharmaceutical therapeutics and compositions provided herein. Treatment encompasses a rotational combinatorial therapy described herein, in which a combination of therapeutics is administered in accord with any regimen described herein.
  • amelioration of the symptoms of a particular disease or disorder by a treatment refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic(s).
  • amelioration of symptoms includes amelioration of side effects of the composition or therapeutic(s).
  • amelioration of adverse symptoms of a disease or disorder is a decrease in number or severity of the symptoms of the disease compared to the number or severity of the symptoms of the disease prior to treatment.
  • amelioration of a disease or condition can include a decrease in levels of a biomarker of the disease, such that the biomarker level is decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the biomarker level prior to treatment.
  • symptoms of the disease are decreased, such as the amount or severity of symptoms that are decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the amount or severity of symptoms level prior to treatment.
  • incidence refers to how often an event occurs.
  • adverse side effect incidence refers to how often adverse side effects occur after administration of a therapeutic, for example the frequency of adverse side effects after administration of a combination therapy provided herein.
  • an adverse side effect(s) can occur 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the time after administration of a rotational combinatorial therapy provided herein.
  • adverse side effects incidence decreases when multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein compared to a monotherapy or compared to a combination that is not rotated (z.e., is continuously administered or is administered over a longer period of time).
  • the incidence of an adverse side effect(s) can decrease 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein, compared to a monotherapy or compared to a combination that is not rotated.
  • severity refers to degree of illness or symptoms of the illness or side effects of the illness or side effects of treatment s) for the illness manifested by a patient.
  • the skilled physician can assess the severity of an illness or progression of the severity of an illness or the severity of side effects of the illness or side effects of treatments with therapeutics for the illness based on the knowledge in the field of medicine and pharmacology.
  • the skilled physician or scientist can assess the adverse side effects associated with administration of therapeutics for a disease, disorder, or condition; for example, as severe e.g., adverse side effects requiring medical care), or as mild (e.g., adverse side effects not requiring medical intervention or treatment).
  • adverse side effects can be classified by severity using medical diagnosis codes used in clinical assessment and tracking, such as classification of severity as minor, moderate, major, and extreme.
  • adverse side effects following administration of the rotational combinatorial therapy provided herein are less severe than adverse side effects following administration of monotherapy or following administration of a combination therapy that is not rotated.
  • “adverse effect” or “adverse side effect” refers to a harmful, deleterious and/or undesired effect of administering a medication or drug.
  • a dose-limiting toxicity refers to the dose of a drug that produces side effects severe enough to prevent larger doses being given. It is within the level of skill of a skilled physician to assign or determine a DLT depending on the treatment protocol, the administered treatment, the disease to be treated, the dosage regime, and the particular patient to be treated. Generally, for the treatments, protocols, and regimens provided herein, a DLT is the dose of a drug results in an adverse event or side effect that on the toxicity scale of at least an ongoing or persistent Grade 2 toxicity that fails to resolve over the course of treatment and that limits the patient’s ability to comply with the protocol therapy. As part of rotational therapy, a drug that results in such side effects can be administered at a lower dose, since the combinations of drugs/treatments can act synergistically, or the drug can be discontinued, and optionally replaced with another drug with similar activity or effect.
  • prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced.
  • Prophylaxis includes reduction in the risk of developing a disease or condition and/or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease and/or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease.
  • Prevention includes inhibition or avoidance of a disease, disorder, or condition by administration of a rotational combination therapy provided herein.
  • a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound, which, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses, such as a rotation of combinations of therapeutics.
  • a prophylactically effective amount can be administered in one or more administrations.
  • a prophylactically effective amount of a combination therapy as described herein can be lower than a prophylactically effective amount of a monotherapy.
  • a prophylactically effective amount of a rotational combinatorial therapy as described herein can be lower than a prophylactically effective amount of a monotherapy or a combination therapy that is not rotated.
  • an “effective amount” of a compound or composition for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount can be administered as a single dosage or can be administered according to a regimen, whereby it is effective. The amount can cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease.
  • a measure of effective amount or dosage is the median effective dose (ED50), which is the dose that produces a response that is fifty percent of the maximum obtainable response.
  • ED50 median effective dose
  • an effective amount of an individual therapeutic or a therapeutic combination is the amount required to achieve a desired amelioration of symptoms.
  • a rotational combination therapy as can employ a lower effective amount of drug compared to a than a monotherapy or a combination therapy that is not rotated.
  • a therapeutically effective amount or a therapeutically effective dose refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting, or partially arresting a symptom of a disease or disorder.
  • a therapeutically effective amount or dose can reference the amount or dose of a particular therapeutic in a combination, and/or can reference the amount or dose of the combination of therapeutics in a combination.
  • therapeutic efficacy refers to the ability of an agent, compound, material, composition, or combination of agents, compounds, materials, or compositions containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, composition, or combination of agents, compounds, materials or compositions containing a compound has been administered.
  • the therapeutic efficacy can refer to the therapeutic efficacy of a combination of therapeutics or a plurality of combinations, such as the combination(s) for use in a rotational combinatorial therapy.
  • a standard dosage of a therapeutic is the dosage for formulation or administration that is approved by applicable regulatory agencies in the field, for example the U.S. Food and Drug Administration (FDA), such as the FDA Data Standards Advisory Board.
  • a standard dosage includes an amount (z.e., mass) of a therapeutic (ie., drug) for formulation.
  • a standard dosage also can include a dosage regimen for administering the standard dosage (z.e., biweekly dosing).
  • a standard dosage as approved by a regulatory agency or body also can be a range of dosages or the dosage can vary based on characteristics of the patient to whom the therapeutic is administered. For example, the dosage can vary depending on the weight of the patient, or the severity of the disease or condition for which the therapeutic(s) is/are administered.
  • a standard dosage also can be defined by pooled analysis using scientific studies of real-world data, where efficacy is established for various doses, and the dosage is established as a dosage with a particular efficacy. When standard dosage is established, efficacy, adverse side effects, and other factors are considered.
  • An individual therapeutic (ie., drug) in a combination for a rotational combinatorial therapy provided herein can be formulated in a standard dosage.
  • an individual therapeutic (z.e., drug) in a combination for a rotational combinatorial therapy provided herein is initially formulated in a standard dosage and the dosage is modified from the standard dosage for subsequent administrations. For example, the dosage of the individual therapeutic is formulated in a lower or higher dosage compared to a standard dosage.
  • polypeptide, peptide, and protein refer to polymers of amino acids of any length. Where not used interchangeably other characteristics, such as size, and structure, are contemplated, as defined below.
  • the polymer can be linear or branched, and can contain amino acids, including modified amino acids, and it can be interrupted by non-amino acids.
  • amino acid polymers that include sequence modifications including, replacements, insertions, deletions, and transpositions.
  • amino acid polymers that contain post-translational modifications, such as disulfide bonds, glycosylation, sialylation, conjugation to other proteins, peptides, and polypeptides, such, but not limited to, conjugation to a detectable marker, or reporter.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4t h Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).
  • Such substitutions can be made in accordance with those set forth as follows:
  • substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.
  • Amino acid substitutions contemplated include conservative substitutions, such as those set forth in the table below. Substitutions that alter properties of the peptides cam be effected; such substitutions are generally non-conservative, but can be readily effected by those of skill in the art. Conservative amino acid substitutions generally can be effected without altering the activity of a peptide. Variants of the weight loss peptides are known and/or can be used herein.
  • a protein is a polypeptide that has a three-dimensional structure and can include bonds in addition to peptide bonds, such as disulfide bonds and other interactions, that participate in forming the two- and three-dimensional structure.
  • a peptide refers to a polypeptide that is from 2 to 300, but generally shorter than 100, amino acids in length.
  • Peptides include therapeutic peptides that are administered for treatment of a disease, disorder, or condition. Peptides that are administered for therapeutic treatment are administered in an amount effective to elicit a therapeutic effect. Peptides for treatment can be administered at a dosage wherein the resulting circulating or accumulating levels of the peptide mimic normal levels or the peptide levels of a person who is not afflicted by the disease or condition.
  • a therapeutic peptide or peptides also can be administered at a dosage so that the circulating level of the peptide is similar or the same of an endogenous peptide that elicits a therapeutic effect.
  • gastric bypass is the most effective treatment to effect sustained and significant weight loss. It is known in the art that gastric bypass, not only reduces the size of the stomach to thereby limit food intake; it also results in changes in peptide hormones involved in regulating food intake and effects (see, e.g., Beckman et al. (2010) J Am Diet Assoc. 110:571-584, doi: 10.1016/j.jada.2009.12.023).
  • peptides or agonists
  • GLP-1 glucagon-like peptide-1
  • PYY peptide tyrosine-tyrosine
  • SEQ ID NO:3 leptin
  • Antagonists of peptides that decrease after bypass can be administered.
  • polypeptide is an amino acid chain that contains a plurality of peptides, and is generally 100 amino acids or longer.
  • polypeptides and peptides can be used interchangeably to refer to the therapeutics, such as those for weight loss.
  • parenteral and parenterally refer to administration of an agent via any route other than oral administration.
  • Parenteral includes the injection of a dosage form into the body by a sterile syringe or some other mechanical device, such as, for example an infusion pump.
  • parenteral administration includes injection (z.e., subcutaneous, intramuscular, and/or intravenous injection), infusion, implantation, intraperitoneal routes of administration, and any other mode of delivery other than ingestion to any site in or on the body of a subject.
  • monotherapy refers to the use of a single therapeutic (z.e., drug or medication) to treat a particular disorder or disease.
  • a monotherapy can include continued treatment, such as for months or years, with a single therapeutic.
  • combination therapy refers to the administration of two or more different therapeutics or other treatment(s), such as radiation and surgery.
  • Multiple therapeutic agents or treatments in the combination therapy can be provided and/or administered separately, sequentially, intermittently, simultaneously, or provided in a single composition.
  • the two or more different therapeutics or other treatment(s) in a combination therapy are administered together or separately, or intermittently.
  • the treatments can be within 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, or 1 week of the other treatments in the combination.
  • the timing and order of administration depends upon the disease, disorder, or condition, and is within the judgement of a physician.
  • an orexigenic pathway is one that promotes appetite. Orexigenic pathways can be activated by increased AMP-activated protein kinase (AMPK) activity by ghrelin to promote appetite (orexia).
  • AMPK AMP-activated protein kinase
  • the peptides orexin A (SEQ ID NO:36), neuropeptide Y (NPY) (SEQ ID NO: 35) and ghrelin (SEQ ID NO:23) stimulate appetite and act through orexigenic pathway(s).
  • a receptor is a molecule that has an affinity for a particular ligand.
  • a receptor is a protein that specifically binds a signal molecule and then initiates a response.
  • Receptors can be naturally-occurring or synthetic molecules. Receptors also can be referred to in the art as anti-ligands. In some examples, multiple receptor types are involved in a signaling pathway. In the context of pharmacology, receptors include macromolecules in the membrane or inside the cell that specifically (chemically) bind a ligand (drug).
  • downregulate or downregulation refers to reducing or suppressing the body’s response to specific stimuli.
  • Receptor downregulation is characterized by a decrease in total receptor number in the cell.
  • Receptor downregulation can be caused by long-term exposure to agonists.
  • Cells undergo receptor downregulation as a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cellular response to that level of exposure.
  • receptor downregulation occurs and can decrease the drug efficacy due.
  • Rotational and/or combinatorial drug therapy described herein decreases receptor downregulation due to the activation of multiple pathways as opposed to the one activated pathways in monotherapy.
  • Rotational and/or combinatorial drug therapy described herein also can overcome the decreased therapeutic response due to receptor downregulation by rotating to a next combination with therapeutics that activate a pathway that is not downregulated.
  • body mass index is a value derived from the mass (weight) and height of a person.
  • the BMI is defined as the body mass divided by the square of the body height, and is expressed in units of kg/m 2 , resulting from mass in kilograms and height in meters.
  • BMI value is used to categorize a person as underweight (under 18.5 kg/m2), normal weight (18.5 to 24.9), overweight (25 to 29.9), or obese (30 or more) based on tissue mass (muscle, fat, and bone) and height.
  • BMI under 20 and over 25 have been associated with higher all-causes mortality, with the risk increasing with distance from the 20-25 range.
  • weight loss means a decrease in body mass of a person. Weight loss includes overall loss of fat, muscle and water compared to a pre-set time point. In examples herein, weight loss is assessed after treatment with a rotational combinatorial therapy compared to weight prior to treatment.
  • off label use means the U.S. Food and Drug Administration (FDA) has approved the drug for a particular use and the approved drug is used for a different therapeutic use.
  • the FDA has determined the benefits of using the drug for a particular use outweigh the potential risks, but has not assessed the risk or benefits of the drug for the off-label use.
  • potency is the drug concentration required to produce an effect of a specified intensity. Potency generally is calculated as the concentration (or dose) required to produce 50% of the drug’s maximal effect (EO50). EC50 is used to express the potency in in vitro studies and also is the dose required for an individual to experience 50% of the maximum effect, and median effective dose or ED50 is used to measure a drug’s potency in a population (z.e., animal studies or human populations). ED50 is the dose that produces the desired effect in 50% of the population. As applied to clinical settings, potency can indicate the dose of the drug, whereas efficacy can indicate the magnitude of the response (regardless of the dose).
  • an “adverse effect,” or “side effect,” or “adverse event,” or “adverse side effect,” refers to a harmful, deleterious and/or undesired effect associated with administering a therapeutic agent.
  • side effects associated with the administration of a monotherapy such as continuous administration of a monotherapy over an extended period of time.
  • Such adverse side effects include, for example, headaches, nausea, diarrhea, heartburn, gas, constipation, dry mouth, dizziness, increased blood pressure, increased heart rate, restlessness, drug dependence, abuse, and withdrawal symptoms.
  • Other serious adverse effects include infections, such as tuberculosis, and other infections caused by viruses, fungi and bacteria, including upper respiratory infections, as well as dermatological and dermal toxicity, such as rash.
  • “adverse effect” or “side effect” refers to a harmful, deleterious and/or undesired effect of administering a therapeutic agent.
  • Side effects or adverse effects are graded on toxicity, and various toxicity scales exist, providing definitions for each grade. Examples of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning grades of severity is within the skill of an experienced physician or other health care professional. The severity of symptoms can be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. The CTCAE is a descriptive terminology used for Adverse Event (AE) reporting.
  • CTCAE Adverse Event
  • the grading (severity) scale is provided for each AE term.
  • the CTCAE displays Grades 1 through 5, with clinical descriptions for severity for each adverse event based on the following general guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Death related to AE/ fatal).
  • serum level refers to the amount of a therapeutic in blood plasma. Serum level can be used to assess whether the amount of the therapeutic administered is safe and/or effective.
  • Therapeutic drug monitoring of the concentration of mediation(s) in body fluids such as blood serum can be used during treatment and/or for diagnostic purposes. Assessing drug serum levels can be used, for example, to avoid drug toxicity, to determine if drug serum level is toxically high; to adjust dose, such as, for example, after reaching a steady state, to determine if the loading dose was adequate, and/or to predict a patient’s dosing requirements; and for monitoring patient compliance, diagnosing undertreatment (ie., when drug dose can be increased), and diagnosing ineffective treatment.
  • comorbidity refers to the presence of one or more additional conditions co-occurring (that is, concomitant or concurrent) with a primary condition. Comorbidity indicates that the one or more conditions occur simultaneously with the primary condition or as a result of the primary condition.
  • hypertension can be a comorbidity of obesity, where a subject has hypertension as a result of the obese state.
  • complications of the primary condition and the comorbidity are the same or overlap.
  • disease or disorder refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms.
  • Diseases and disorders of interest herein are those that can be treated with a rotation of pharmaceutical agents.
  • Diseases and disorders also include those where treatment of the disease or disorder is impaired due to downregulation of receptors or drug targets, leading to a decreased efficacy of the therapeutics.
  • Of particular interest herein are those disorders where treatment is not effected due to resistance to the expected efficacy of the therapeutic molecule or drug or peptide.
  • chronic is used to characterize the duration of a disease, disorder, or condition.
  • a chronic condition is a condition that is prolonged and requires ongoing medical intervention or limit activities of daily living or both.
  • a chronic condition generally is a condition that lasts more than 3 months, 6 months, 1 year or more, or that is terminal.
  • a chronic condition can persist when the affected individual receives treatment or therapeutics, and during the amelioration of signs or symptoms of the condition.
  • Chronic conditions include chronic diseases or disorders.
  • Chronic conditions can be characterized by one or more of etiology, duration, onset, recurrence/pattem, prognosis, sequelae, diagnosis, severity and prevalence (see e.g., O’Halloran et al., Family Practice, (2004) 21(4): 381-386).
  • obesity refers to a condition in which the subject has abnormal or excessive fat accumulation, such as a body mass index of greater than 30.
  • Obesity can be a health risk.
  • Obesity can be caused by a multitude of factors, including genetic and environmental factors.
  • Patients with obesity can have other comorbidities, such as, for example, high blood pressure, type 2 diabetes, cardiovascular disease, high cholesterol, and others.
  • overweight refers to a condition in which the subject has extra fat accumulation or weight that is higher than typical, with a body mass index that is, for example, greater than 25. Overweight also can be assessed by other methods known in the art, such as by assessing waist and/or hip circumference, subcutaneous fat thickness, percent of fat or muscle compared to total body composition, overall body weight, and similar assessments. Other metrics can be used to determine whether a subject is overweight, such as a waist to hip ratio of greater than, for example, 1.0. Overweight subjects are at increased risk various comorbidities, including high blood pressure, high cholesterol, COPD, type II diabetes, and others. Overweight can be caused by a multitude of factors, including genetic and environmental factors.
  • a “pharmacokinetic property” refers to a property related to the action of a drug or agent, such as a therapeutic peptide, in the body and in particular the rate at which drugs are absorbed, distributed, metabolized, and eliminated by the body.
  • Pharmacokinetics can be assessed by various parameters. These include, but are not limited to, clearance, volume of distribution and serum half-life.
  • Pharmacokinetic properties of peptides can be assessed using methods well known in the art, such as, for example, administering the peptide to a human or animal model and assessing the amount of the peptide in the body (e.g., in the bloodstream) at various time points.
  • the various parameters, such as clearance, volume of distribution and serum half-life are assessed using calculations well known in the art and described herein.
  • “improved pharmacokinetic properties” refers to a desirable change in a pharmacokinetic property of a peptide or combination of peptides or combination of therapeutics, such as peptides in a pharmaceutical composition for administration with the timing or regimen set forth herein, compared to, for example, a peptide or therapeutic administered continuously or once.
  • the change can be an increase or a decrease.
  • “synergistic effect” or “synergy” or grammatical versions thereof refers to a larger therapeutic effect of the combined treatment compared to the effect predicted from the sum of each therapeutic alone. For example, when the action of one drug is increased when administered in the presence of another drug.
  • plasma half-life refers the elimination half-life of a peptide(s) or therapeutic(s) or combinations thereof or the time at which the plasma concentration of the administered peptide(s) or therapeutic(s) or combinations thereof has reached one half of its initial or maximal concentration following administration.
  • Reference to plasma half-life includes plasma half-life during the a-, P-, and/or y- phase. Plasma half-life can be assessed using methods well known in the art.
  • assays in which a peptide(s) or therapeutic(s) or combinations thereof is administered to subjects can be performed, and the plasma half-life of the peptide(s) or therapeutic(s) or combinations thereof assessed by measuring the amount of the peptide(s) or therapeutic(s) or combinations thereof in the plasma at various time points.
  • the t’AB for example, is calculated as -ln2 divided by the negative slope during the terminal phase of the log-linear plot of the plasma concentration-versus- time curve.
  • the plasma half-life can aid the skilled artisan in determining when a secondary peptide or therapeutic or combinations thereof can be administered to a subject, such as, for example, in a rotational therapy regimen described herein.
  • clearance refers to the removal of an agent, such as a peptide, from the body of a subject following administration. Clearance can be assessed using methods well known in the art. For example, assessment of peptide levels in blood or serum or another fluid from a patient administered a peptide or combination of peptides or therapeutics can be performed, and the clearance of the peptides from the body assessed by measuring the amount of the peptide(s) in the plasma at various time points and calculating the clearance as Dose / AUC 0-inf. In some examples, clearance of a peptide results in a decrease in the circulating peptide levels in serum compared to circulating levels directly or recently after peptide administration.
  • the clearance of administered peptide(s) or therapeutic(s) or combinations thereof can result in a decrease in circulating levels by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to circulating levels directly or recently after peptide administration.
  • the term assess or assesses and grammatical variations thereof is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a pharmaceutical(s) or therapeutic(s) or phenotype(s), and of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity.
  • Assessment can be direct or indirect. For example, assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein on a subject for weight loss can be evaluated by a variety of metrics, including fat, weight, water, and muscle loss or gain. Assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein also can be evaluated by measuring levels of the therapeutics or other molecules in the bloodstream.
  • a decrease in the amount of the therapeutic(s) such as, for example, a therapeutic peptide or combinations of peptides in the bloodstream or serum can indicate that another peptide(s) or therapeutic(s) or combinations thereof can be administered to the subject.
  • a secondary therapeutic(s) z.e., peptide e.g., therapeutic peptide
  • peptide(s) e.g., therapeutic peptide(s) can be administered when the concentration of the primary is decreased by at least or about or about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the level or amount of the therapeutic(s) directly after administration, where the level of amount of the therapeutic(s) directly after administration is assessed at least or at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3
  • subject refers to an animal, including a mammal, such as a human being. Subjects include human patients.
  • a “patient” refers to a human subject.
  • “patients” or “subjects” are humans who participated in a therapeutic regimen, such as a therapeutic regimen described in the examples herein.
  • a “patient” is a human who has a chronic condition, disorder, or disease, such as overweight or obesity. Patients or subjects can be treated with the rotational combinatorial therapy herein.
  • animal includes any animal, such as, but not limited to, primates including humans, gorillas, and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs; and other animals.
  • rodents such as mice and rats
  • fowl such as chickens
  • ruminants such as goats, cows, deer, and sheep
  • pigs and other animals.
  • Non-human animals exclude humans as the contemplated animal.
  • a “control” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest.
  • a control also can be a subject, such as a subject that is not treated with rotational combinatorial therapy provided herein, or is treated with a monotherapy, or combination therapy that is not rotated, or a placebo.
  • a control also can be an internal control.
  • composition refers to any mixture of two or more products or compounds, for example, but not limited to, peptides, therapeutic molecules, agents, modulators, and regulators.
  • a composition can be, for example, a solution, a suspension, an emulsion, a liquid, a powder, a paste, aqueous or non-aqueous formulations, and any combination thereof.
  • an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass peptides or drugs, or therapeutics and combinations thereof contained in articles of packaging.
  • Fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
  • kits refers to a packaged combination, optionally including reagents and other products and/or components for practicing methods using the elements of the combination.
  • kits containing a combination of pharmaceuticals provided herein and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of a biological activity or property are provided. Kits optionally include instructions for use.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence, “about 5 hours” means “about 5 hours” and also “5 hours.”
  • an optionally included therapeutic means that the therapeutic is included or is not included.
  • delivery vehicles that display peptides for weight loss and anabolic peptides.
  • delivery vehicles such as liposomes, exosomes, extracellular vesicles, lipid nanoparticles (LPNs), for displaying or containing peptides for treating obesity and/or diabetes, and optionally containing small molecule drugs in or on the vehicles.
  • LPNs lipid nanoparticles
  • proglucagon derived peptides are provided, and anabolic peptides and other peptides and drugs that that promotes muscle growth and/or prevents or inhibits muscle wasting are described.
  • the delivery vehicles display at least one of the proglucagon peptides and a muscle wasting/promoting peptide or a peptide that inhibits muscle loss, such as a myostatin pathway inhibitor, or a plurality thereof.
  • the delivery vehicles display one the peptides and combinations of at least three different delivery vehicles are administered together, such as in a co-formulation, or administered sequentially, or simultaneously.
  • the delivery vehicles are formulated for administration by injection, such as intravenous injection or subcutaneous injection.
  • the delivery vehicles also can be formulated for mucosal administration via contacting with oral mucosa, intestinal mucosa, and/or intranasal mucosa.
  • the delivery vehicles include, but are not limited to, liposomes, such as those exemplified herein, or any suitable liposome, or a lipid nanoparticle (LNP), or an extracellular vesicle, including an animal or plant extracellular exosome, such as a milk-derived, or a microalgal extracellular vesicle (see, International PCT Publication No. WO2023/144127; International PCT application No. WO2023/076418;and International PCT application No. WO2023/076418).
  • the peptides drugs can be displayed on or contained in or both using any suitable delivery vehicle, including extracellular vesicles, exosomes, LPNs, and liposomes.
  • small molecule drugs such as phentermine
  • the delivery vehicle such as introducing it into extracellular vehicle or fabricating them together.
  • delivery vehicles are provided that deliver combinations of drugs to treat obesity and reduce or eliminate the concomitant muscle loss that accompanies weight loss by enhancing muscle growth.
  • Suitable peptides and combinations thereof and regimens are described herein and in commonly owned International PCT application No. PCT/US23/77508 (now subsequently published International PCT publication No. WO2024/091863).
  • combinatorial treatment protocols and rotational combinatorial treatment protocols in which diseases, disorders, and conditions are treated by targeting a plurality of molecules and/or pathways involved in the disease, disorder, or condition, to reduce or avoid desensitization to a particular treatment, and also can reduce toxicity and adverse side effects.
  • the diseases, disorders, and conditions include obesity, diabetes, and associated diseases, disorders, and conditions, such as obesity comorbidities.
  • the combination of treatments can be rotated so that a subject is treated with different combinations of treatments for limited periods of time.
  • methods for developing such protocols including selecting a disease, disorder, or condition for treatment with a combinatorial protocol and/or a rotational combinatorial protocol.
  • These protocols and methods are exemplified herein with respect to obesity, and several other diseases, disorders, and conditions that are difficult to treat and that become resistant to treatment over time.
  • the peptides and other drugs are displayed on or provided in delivery vehicles, such as liposomes and exosomes, and formulated for administration to a subject, such as by injection, including subcutaneous injection, such as by autoinjector.
  • Obesity is exemplar of a disease, disorder, or condition, as shown herein that can be treated with a combinatorial, particularly a rotational combinatorial treatment protocol.
  • Obesity is a medical condition in which excess body fat has accumulated. The excess body fat has adverse effects on health (see, e.g., “Obesity and overweight Fact sheet N°311,” published by the World Health Organization (WHO) in January 2015. Retrieved 2 February 2016). Obesity increases the likelihood of various diseases and physical and mental conditions.
  • these increases are manifested in metabolic syndrome (Haslam etal., (2005) Lancet 366 (9492): 1197- 209), and include cardiovascular diseases, high blood pressure, high blood cholesterol, high triglyceride levels, diabetes mellitus type 2, obstructive sleep apnea, certain types of cancer, osteoarthritis, and depression (Haslam et al., (2005) Lancet 366 (9492): 1197— 209). Obesity reduces life expectancy. (Jura etal., (2016) Age (Dordr) 38(1):23; Peeters etal., Ann lnternMed (2003) 138(l):24-32).
  • the biochemical link between the development of obesity and health consequences fall into two broad categories: those attributable to the effects of increased fat mass, such as osteoarthritis, obstructive sleep apnea, and social stigmatization, and those due to the increased number of fat cells, such as diabetes, cancer, cardiovascular disease, non-alcoholic fatty liver disease (Stenkula etal., Am J Physiol Regul Integr Comp Physiol. (2016) 315(2):R284-R295).
  • Increases in body fat alter the body's response to insulin, potentially leading to insulin resistance.
  • Increased fat also creates a proinflammatory state and a prothrombotic state (Kawai et al., Am J Physiol Cell Physiol.
  • Combinatorial protocols involve the administration of a plurality of treatments, each of which targets different pathways involved in the disease, disorder, or condition.
  • the combinatorial protocols involve combination of at least three different treatments, such as at least two peptides that promote or result in weight loss and one that prevents or reduces muscle loss associated with weight loss.
  • the combinatorial protocol(s) can be integrated into a rotational combinatorial protocol in which different combinations of treatment are rotated. Rotational protocols can be selected where monotherapy or combination of treatments become less effective or require higher doses to be effective.
  • Provided herein are exemplary rotational combinatorial protocols and methods of developing such protocols.
  • a delivery vehicle such as an exosome or extracellular vesicle or LPN.
  • Liposomes are exemplary of such vehicles.
  • the peptide drugs can be displayed on the vehicle or incorporated into it; the small molecule drugs can be incorporated or associated with the surface so that they can be delivered together.
  • the stoichiometry of the amounts of each drug can be adjusted to deliver the proper dosage, adjust, and provide relative amounts or ratios of the drugs for dosing the proper amount of each.
  • the platform provides the ability to adjust various parameters and deliver combinations of drugs for treating obesity and concomitantly avoiding or reducing muscle loss that accompanies weight loss.
  • The can be provided in or on separate vehicles, which can be mixed before administration to provide combinations.
  • the delivery vehicles such as liposomes
  • each of the unique combinations can be rotated, such as every three months to prevent drug desensitization and reduce or avoid adverse side effects.
  • the peptide stoichiometry can be adjusted, such as by modifying the surface of the liposome to reflect a dominant population of the more efficacious peptide or to increase the dose of one or more relative to others.
  • the size of the delivery vehicle can be adjusted or selected to affect the amount of drug(s) in or on the liposome and to control binding kinetics of the surface peptides.
  • Liposome are among the delivery vehicles. Liposomes and other vehicles provide several advanced pharmacokinetic features, including: a. protection of the peptides from endogenous peptidases in serum, thus extending half life b. a delivery system within the liposome itself, of which we will describe below.
  • combinational, combinatorial and rotational therapies provide advantages compared to monotherapy. They permits treatment via multiple pathways or targets involved in obesity or other disease, disorder, or condition.
  • the delivery vehicles also can for fabricated or formulated to deliver small molecule drugs including, but are not limited to: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, in addition to the peptides.
  • drugs including, but are not limited to: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Dieth
  • the combinations thus, include at least two or three known peptides for treatment of obesity and additionally one or more peptides (or other drugs) that reduce or eliminates muscle loss that accompanies weight loss.
  • the drugs that reduce or eliminate muscle loss re muscle enhancing anabolic peptides such as sermorelin, tesamorelin, IGF-1, or other growth hormone or peptide derived therefrom.
  • Other drugs that inhibit the causes of muscle loss also are known, and, optionally, can be included in the combinations. These include myostatin pathway inhibitors, such as the antibody bimagrumab, which inhibit the activity of myostatin.
  • Myostatin also is known as growth differentiation factor 8 or GDF-8; it is a member of the transforming growth factor-P (TGF-P) superfamily.
  • TGF-P transforming growth factor-P
  • Myostatin is a negative regulator of skeletal muscle growth, so its inhibition can contribute preservation of lean muscle mass. Inhibitors of myostatin, thus, are distinct from muscle enhancers promote muscle growth; they have a different target and mode of action. Myostatin inhibitors are not muscle enhancers. It has been reported that myostatin pathway inhibitors used in combination with a GLP-1 pathway agonist can enhance overall weight loss (see, e.g., US patent application publication US 20240368262).
  • delivery vehicles such as liposomes, provided herein include, but are not limited to:
  • the delivery vehicles can provide “dual peak” activity. They display peptides, which can have an immediate pharmacological effect, and then, once the vehicle, such as the liposome ruptures, there is a second time release of any weight loss push. There can be a further peak or release of any drug/peptide from the ruptured vehicle .
  • serum levels and other parameters can be calculated easily from known prescribing doses (for example, phentermine is prescribed at 37.5mg daily) and then packaged into each vehicle in an amount to mimic that to achieve therapeutic serum levels and match other parameters.
  • the delivery vehicles can concurrently deliver the small molecule drugs to treat other diseases, such as diabetes (metformin), hypertension (ACEi/ARBS), dyslipidemia (Statins), and others for obesity co-morbidities.
  • diseases such as diabetes (metformin), hypertension (ACEi/ARBS), dyslipidemia (Statins), and others for obesity co-morbidities.
  • auto-injectors for selfadministration are routinely used for administration. Volumes up to 3 mL generally are administered. Higher volumes can be administered.
  • high speed autoinjectors can deliver higher doses, as can adding excipients, such as a hyaluronidase, to the compositions.
  • the high speed autoinjectors inject fairly large volumes quickly, and the formulations also contain excipients, such as a soluble hyaluronidase, to facilitate administration of large volumes
  • Typical GLP-l/GIP injectors for example, deliver from 0.5ml to 3ml.
  • small molecule drugs such as phentermine can be incorporated into or onto the liposomes.
  • Small molecules inside the liposome or other vehicle provide unique pharmacokinetic properties.
  • the peptides act immediately since they are on the outside of the liposome and actuate a chain of molecular events for weight loss synergistically (First phase).
  • Second phase As the liposome dissolves, there are some small molecules inside the liposome that are then released (2nd Phase), and eventually any small molecules attached to the inner surface of the liposome will be available for binding to its receptor (third phase).
  • the vehicles are liposomes or exosomes that contain at least 2, generally at least three, peptide drugs, and additionally muscle enhancer, such as an anabolic hormone.
  • peptide drugs there are at least 12 different peptides, which are known to produce or facilitate weight loss, and at 2 or 3 anabolic peptides, exemplified herein.
  • GLP- 1 SEQ ID NO: 1
  • Leptin SEQ ID NO:3
  • Oxyntomodulin OXM
  • SEQ ID NO:4 PYY (SEQ ID NO: 6), Amylin (SEQ ID NO: 7); Tesamorelin (SEQ ID NO: 8), GIP, SEQ ID NO: 10; CCK (SEQ ID NO: 11); Glucagon (SEQ ID NO:27); Sermorelin (SEQ ID NO:5); and IGF1 (SEQ ID NO:44), and variants or biologically active portions of each as well known in the art and/or detailed herein.
  • Other known peptides and small molecule weight loss drugs can be included.
  • Drugl GLPl/GIPl/Oxyntomodulin + GH (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 4 or 47 + SEQ ID Nos: 5, 8, 44
  • Drug2 GLPl/GIPl/Amylin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 7 + SEQ ID Nos: 5, 8, 44
  • Drug3 GLP1/GIP1 /Glucagon + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 27 + SEQ ID Nos: 5, 8, 44
  • Drug4 GLP1/GIP1/CCK+ GH (Sermorelin or Tesamorelin or IGF1)
  • Drug5 GLP1/GIP1/PYY + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 6 or 37 + SEQ ID Nos: 5, 8, 44
  • Drug6 GLP1/GIP1 /Leptin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 3 + SEQ ID Nos: 5, 8, 44.
  • the polypeptides can be pegylated or modified by group, such azido for click chemistry.
  • Delivery vehicles are prepared by known methods, including those exemplified herein, for linkage of the polypeptides.
  • Lysine (K) residues for which pegylation reduces or eliminate activity of the peptide when displayed, are replaced with a conservative amino acid replacement, such as Arg, Glu, Gin, His, so that only a lysine or lysine residues that do not substantially alter activity are available for pegylation.
  • Peptides can be linked to pre-formed liposomes or linked to components of the liposomes during preparation of the liposomes.
  • a goal is to preserve as much of the tertiary conformation of the native peptides need to retain activity, reduce steric hinderance between or among the up to 4 peptides on a single liposome, protect the N-terminus from serum proteases, and/or, for example, set up an azio-PEG24 linker for copper- catalyzed azide-alkyne cycloaddition (CuAAC) and bioconjugation to preformed liposomes.
  • CuAAC copper- catalyzed azide-alkyne cycloaddition
  • the stoichiometry can be assessed, for example, in vitro via cAMP assays as described below.
  • liposomes are fabricated as exemplified.
  • the peptides can be acetylated at the N-terminus to protect them from in vivo degradation.
  • the activity of the peptides displayed on the resulting delivery vehicles can be assessed by known assays, including commercially available kits. These kits are based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenous labeled cAMP conjugate that acts as a competitor. This is followed by detection of the labeled cAMP conjugate using a variety of detection technologies, including fluorescence resonance energy transfer (FRET) or enzymatic reactions.
  • FRET fluorescence resonance energy transfer
  • ADSCs Adipose-derived stem cells
  • the peptides are provided linked to or incorporated into the surface of delivery vehicle.
  • the following sections describe that the vehicles contain three components or aspects: 1) the delivery vehicle; 2) linkers or linkages; 3) the peptides.
  • Peptides listed herein can be incorporated in and/or on the delivery vehicles.
  • proglucagon-derived peptide PGDP
  • Bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1/3 and PC2 ( Figure 1).
  • PGDP peptides exert unique physiological effects that influence metabolism and energy regulation; several of these peptides have been exploited in the form of long-acting, enzymatically resistant analogues for treatment of various pathologies. These can be linked to delivery vehicles (or incorporated in the surface thereof) as described herein for use in combination with other such peptides and/or delivery vehicles.
  • Proglucagon is expressed in both alpha-cells of the pancreatic islets as well as neuroendocrine L-cells primarily located in the distal ileum and colon.
  • the PGDP profile is not identical in the pancreas and gut, due to differential post- translational processing of proglucagon by tissue-specific enzymes termed prohormone convertases (PC).
  • PC prohormone convertases
  • pancreatic alpha-cells mainly possess PC2, which cleaves proglucagon to generate glicentin-related pancreatic peptide (GRPP), glucagon, intervening peptide- 1 (IP-1) and major proglucagon fragment (MPGF).
  • GRPP glicentin-related pancreatic peptide
  • IP-1 intervening peptide- 1
  • MPGF major proglucagon fragment
  • proglucagon is cleaved by PC 1/3 yielding glicentin, GRPP, oxyntomodulin (OXM), GLP-1, intervening peptide-2 (IP- 2) and GLP-2. Some degree of crossover exists.
  • the gut and brain are extra-pancreatic sources of glucagon, while local intraislet GLP-1 production has also been established in alpha cells, particularly in times of beta-cell stress.
  • the solitary nucleus of the medulla oblongata which utilizes PC 1/3 in a similar fashion to the gut generates PGDP’s in the central nervous system (CNS) b.
  • CNS central nervous system
  • SM skeletal muscle
  • a goal is to provide regimens that mimic secretory and pulsatile response of these endogenous peptides that occur following RYGBP and other bariatric weight loss surgeries.
  • delivery vehicles for effecting and providing the combination therapies.
  • Combinations of peptides are described throughout the disclosure herein. It is to be understood that the mixtures of peptide therapeutics can be linked to or incorporated into a delivery vehicle, such as a liposome or extracellular vehicle, such as an exosome, or a synthetic lipid-based nanoparticle.
  • the delivery vehicles can be prepared to deliver a single peptide or a plurality thereof, mixed to provide complementary activities to mimic hormonal and other changes that occur following bariatric surgery.
  • the general approach is to administer one or two peptides for fat/weight loss and one or more to prevent or inhibit muscle loss associated with fat loss or weight loss, or a peptide that promotes muscle growth.
  • two or three proglucagon peptides can be linked to a liposome, and a peptide that inhibits or prevents muscle wasting or that enhances muscle production can be prepared.
  • few than all of the peptides can be linked to a liposome, and mixtures of liposomes delivering the combination of peptides can be co-formulated or administered at the same time or serially.
  • Included among the peptides for linkage to or conjugation to or incorporation into a delivery vehicle, such as a liposome are the following:
  • PeptideFL2 Adiponectin
  • PeptideFL4 Oxyntomodulin
  • PeptideFL5 PYY
  • PeptideFL6 Amylin
  • PeptideFL7 Pancreatic peptide
  • PeptideFL8 Enterostatin/GIP (Gastroinhibitory Polypeptide)
  • PeptideFLl 2 HGH 176-191
  • PeptideFLl 4 Neurotensin
  • PeptideFLl 7 MPGF (major proglucagon fragment)
  • Delivery vehicles include any lipid nanoparticles on which peptides can be displayed on the surface and that are biologically compatible so that they can be administered to a human subject.
  • Delivery vehicles include, but are not limited to, lipid nanoparticles, extracellular vesicles, liposomes, and other such delivery vehicles known to those of skill in the art.
  • Exemplary of lipid-based delivery vehicles are exosomes, such as extracellular delivery vehicles, and liposomes.
  • the peptides can be incorporated into the surface of the vehicles, such as by recombinant expression in a surface protein, or by chemical linkage to a moiety on the surface.
  • the surface can be modified for linking peptides by methods known to those of skill in the art, including those described herein.
  • Liposomes are exemplary delivery vehicles
  • Liposomes are versatile drug delivery vehicles. They have unique properties, which include, for example, site-targeting, sustained or controlled release, protection of drugs from degradation and clearance, superior therapeutic effects, and lower toxic side effects. Liposomes include large multilamellar vesicles (LMV), and small unilamellar vesicles (SUV). Lamellas are phospholipid bilayers, in which the fatty acids are inside the bilayer, and polar head groups are outside in contact with water. LMVs can have diameters in micrometer range, while SUVs can be classified as nanoparticles, because diameter is typically under 100 nm. SUVs can traffic in vivo in the blood and go anywhere blood goes.
  • LMV large multilamellar vesicles
  • SUVs small unilamellar vesicles
  • Liposome preparation include any of the following techniques, assuming the smallest possible particle to effectively carry at least 3 or 4 total (2 o3 fat loss (FL) peptides, 1 muscle enhancing (ME) peptide peptide) peptides without causing steric hinderance for each of their respective targets. Film hydration methods, double emulsification method, solvent injection techniques, sonification, and in situ preparation of liposomes are well-established methods for preparing the liposome.
  • size reduction techniques as briefly described are employed. This includes, for example, (ultra)sonication either by bath or probe, French press Barenholtz, extrusion, homogenization, or combination methods, such as freeze-thaw extrusion, freeze-thaw sonication, and a high-pressure homogenization-extrusion technique [Pupo], Among these techniques, extrusion, and high-pressure homogenization (HPH) are among the most frequently employed in pharmaceutical manufacturing.
  • Liposomes can be prepared using phospholipids, such as phosphatidyl choline (PC), ethanol amine (PE), and serine (PS), or phosphatidic acid (PA).
  • Phospholipids include those of natural origin, such as egg yolk, and also cholesterol, such as synthetic cholesterol, Cholesterol, which makes the bilayer tighter and less permeable, can be added.
  • the molar percentage of cholesterol should be less than 60%, 50%, 40% or less.
  • a volatile solvent such as chloroform, methanol, t-butanol, cyclohexane, or a mixture thereof, so that components are evenly distributed.
  • the Concentration of the phospholipids and other component can be low, such as less than 30 mg/ml, less than or equal to 20 mg/ml, less than 15 mg/ml or lower. Solvent then is evaporated, for example, by a rotary evaporator. For in vivo applications the solvent should be completely removed by keeping the lipid mixture under high vacuum for several hours. Dried lipid mixture is hydrated, such as with physiological salt solution (0.9 % NaCl), or other suitable buffer. Hydration can take several hours. At this point phospholipids are in thick multilamellar film or cake. Mechanical mixing leads to the formation of the LMVs. More powerful methods such as sonification, or high pressure microfluidic spraying break LMVs into SUVs. If hydration solution contains some drug molecules they are partially incorporated inside liposomes. For purposes herein, the biologically active peptides are conjugated to the outer surface of the liposomes.
  • Exemplary groups include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne.
  • Amino groups form amide bond with an activated carboxylic ester, such as N-hydroxy succinimide (NHS; see. e.g., Redford etal., (1991) Cholesterylsuccinyl-N-hydroxysuccinimide as a cross linking agent for the attachment of protein to liposomes, Biochem Pharmacol. 77:307)).
  • NHS N-hydroxy succinimide
  • Thiols bind with maleimide, bromo- or iodo acetyl, pyridyldithio groups.
  • Amines form an amide bond with carboxylate in the presence of water soluble carbodiimide (EDS), and N-hydroxy succinimide (NHS).
  • pegylation is effect whereby lysines in the active are not pegylated.
  • a method for preparing peptides in which selected lysines are pegylated is described below. This is effected by PEGylating the lysine residue prior to synthesis and employing the pegylated residue during peptide synthesis. This method can be used in general for preparing pegylated peptides and polypeptides.
  • Hydrazides bind with carbonyl groups.
  • P-Nitrocarbonate reacts with amines forming an amide bond.
  • Azide and alkyne groups bind with each other very fast in the presence of a copper ion catalyst (click chemistry).
  • Site specific attachment of PEG can be achieved using a transglutaminase enzyme to couple succinimide activated PEG to glutamine.
  • Spacers can be included between the liposome, and peptide. Spacers allow peptides to orientate better for the binding with the receptor. Exemplary of spacers are water soluble polymer chains. For example, polyethylene glycol (PEG) is a commonly used spacer. Oligonucleotides also can be used as spacers. Oligonucleotides allow specific binding of selected peptides so that the amount of peptides can be better controlled. To employ oligonucleotides as spacers, first, selected oligonucleotides are bound with liposomes.
  • oligonucleotides are bound in the same ratio as they are in the reaction mixture.
  • the actual ratio of oligonucleotides can be measured by using a set of complementary oligonucleotides that are conjugated with fluorescent labels. If the ratio of oligonucleotides on the liposomes is equal to the desired ratio, the liposomes can be used as such. If correction is need, some additional oligonucleotides can be used.
  • Peptides are conjugated with complementary oligonucleotides and mixed with the liposomes in a hybridization buffer, whereby that peptides are bound in a desired ratio.
  • Somewhat different methods of attaching peptides on the liposomes employ biotin-streptavidin binding.
  • One streptavidin has four biotin binding sites.
  • One, two, or even three can be used to bind streptavidin with a liposome, and at least one is still available for the binding of peptides.
  • PEG spacers can again be used.
  • On one end is attached biotin, and the other end can be NHS active ester that easily forms an amide bond with PE, when pH is right (slightly basic).
  • the liposomes can be coated with a monolayer of streptavidin.
  • Peptides similarly can be functionalized with biotin-PEG- NHS. The final product is obtained by mixing these peptide derivatives with streptavidin liposomes.
  • Oligonucleotide spacers for attaching peptides also are compatible with streptavidin liposomes.
  • PEG Polyethylene glycol
  • PEG derivatives are used in covalent attachment (z.e., "PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16: 157-82, 1995).
  • PEG has been attached to low molecular weight, relatively hydrophobic drugs to enhance solubility, reduce toxicity and alter biodistribution.
  • PEGylated drugs are injected as solutions.
  • reagents for PEGylation include, but are not limited to, N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3 -hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotri azole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched m
  • NHS N-hydroxysuccinimidyl
  • the polyethylene glycol has a molecular weight ranging from about 3 kD to about 50 kD, and typically from about 5 kD to about 30 kD.
  • Covalent attachment of the PEG to the drug can be accomplished by known chemical synthesis techniques.
  • the PEGylation of protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions.
  • the attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines also can react (e.g., the N-terminal alpha-amine or the ring amines of histidine) if local conditions are favorable. A more directed attachment is possible in proteins containing a single lysine or cysteine.
  • the latter residue can be targeted by PEG-maleimide for thiol-specific modification.
  • PEG hydrazide can be reacted with a periodate peptide and reduced in the presence of NaCNBEE.
  • PEGylated CMP sugars can be reacted with a peptide in the presence of appropriate glycosyl-transferases.
  • One such technique is the “PEGylation” technique where a number of polymeric molecules are coupled to the peptide.
  • the immune system has difficulties in recognizing the epitopes on the peptide's surface responsible for the formation of antibodies, thereby reducing the immune response.
  • the typical potential immune response is an IgG and/or IgM response, while peptides which are inhaled through the respiratory system (i.e., industrial peptide) potentially can cause an IgE response (i.e., allergic response).
  • the polymeric molecule(s) can shield epitope) on the surface of the peptide responsible for the immune response leading to antibody formation. The heavier the conjugate is, the more reduced immune response is obtained.
  • PEG moieties are conjugated, via covalent attachment, to the peptides.
  • Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g., Roberts et al., Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see e.g., Guiotto et al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (see e.g., Chapman et al., Nature Biotech.
  • Succinimidyl PEGs (as above) comprising either linear or branched PEGs can be conjugated peptides.
  • PEGylated peptides have been generated using NHS chemistries, as well as carbonates, and aldehydes, using each of the following reagents: mPEG2-NHS-40K branched, mPEG-NHS-lOK branched, mPEG-NHS-20K branched, mPEG2-NHS- 60K branched; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB- 20K, mPEG-SMB-30K; mPEG-butyraldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin.
  • PEGylated peptides have been prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including peptides PEGylated with Dowpharma's p-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30 kDa). Schemes for effecting PEGylation are well known to those of skill in the art. b. Synthesis of Pegylated peptides for conjugation to liposomes
  • peptides are chemically coupled to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the polypeptides can be Pegylated to provide a group for conjugation to a liposome.
  • One end of the PEG moiety is linked to the peptide, and the other end includes a group for conjugation to the liposome.
  • One or more different peptides can be conjugated with one liposome.
  • PEG is conjugated with an amino group. Amino group can be terminal or in a lysine residue.
  • conjugation can reduce or inhibit peptide activity. It is important to select the site for PEGylation so that activity of the peptide is not altered. Detailed herein is a method to achieve this, such that polypeptide is synthesized with PEGylated lysine(s) at the site(s) desired for PEGylation, and any other lysines have conventional protective groups.
  • the peptide is synthesized in PEGylated form.
  • the lysines are PEGylated, and employed during synthesis of the polypeptide, such as by solid phase synthesis so that a PEG-lysine is incorporated in the peptide chain at a selected lysine residue.
  • Figure 5A schematically depicts a short segment of a polypeptide. Amino acids k, 1, and m are lysines, and that can be separated from each other by several amino acids. In accord with normal peptide synthesis schemes, the terminal carboxylic group is protected by a methyl group that is removed by basic hydrolysis before the next step.
  • the methyl ester of next amino acid is added together with a condensing agent that often is dicyclohexyl carbodiimide (DCCI).
  • DCCI dicyclohexyl carbodiimide
  • the polypeptide often is synthesized on a solid phase surface onto which the growing peptide chain is chemically attached. This allows the washing of the unused reagents and soluble reaction products off.
  • Figure 5B shows a product, in which lysine, residue 1, is PEGylated, and the PEG has an azide group at the end.
  • Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry).
  • PEG-lysines can be added either in amino or carboxylic end of the polypeptide. This kind of addition can be done to make the binding of the polypeptide with liposome stronger.
  • Protective groups include allyloxycarbonyl and trityl groups. These are orthogonal to other protective groups.
  • the epsilon-amino group of lysine is reacted with carboxyl terminated PEG. The bonding is effected via an amide bond that renders the amino group totally inert. As a result, a peptide that contains a PEG moiety in a desired site is produced.
  • the other end of PEG can have a functional group that can be used for the conjugation with a liposome.
  • the functional group can be acetylene that can be reacted with azide that is in the liposome (click chemistry).
  • other lysines can have conventional protective groups.
  • the peptide is synthesized in PEGylated form.
  • Figure 5B depicts a product, in which lysine 1 is PEGylated, and the PEG has an azide group at the end.
  • Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry).
  • Figure 5C shows a PEGylated lysine.
  • FIGS 2A and 2B depicts a liposome 201 (only the outer atomic layer is schematically shown).
  • the liposome contains 1 - 5 % phosphatidyl ethanolamine that has two fatty acids containing at least 16 carbon atoms, generally 18 - 24 carbon atoms each.
  • a PEG spacer 202 having 10 - 200 ethylene glycol moi eties can bound to each amino group.
  • liposomes have one biotin moiety chemically bound per at least 20, 50, 100, or 120, generally about 100 phospholipids.
  • a calculated amount of streptavidin 203 is added. Streptavidin can bind four biotins very strongly; only one or two of these binding sites should be used to bind streptavidin with a liposome, so that two or three binding sites can be used to bind the peptides 205 - 207 (Fig. 2B).
  • the PEG can contain an N-hydroxy succinimide active ester on one end (Fig. 3), and biotin moiety on the other end.
  • Reagents are chosen so that they are water soluble, somewhat stable in water, and react at room temperature.
  • Peptides have also amino groups, and PEG spacers can be connected to these amino groups, such as one PEG spacer 204 having a biotin moiety on the other end. Additional PEGs 208 can be added subsequently to provide stealth property against the body’s immune system that tends to remove foreign particles.
  • Streptavidin coated liposomes provide certain advantages. For example, once liposomes and PEG-peptides are prepared, any and all combinations can be fabricated by mixing the components. The skilled person, such as the physician, can personalize the treatment for the needs and particulars of the treated subject. Personalized regimens of combinations of the therapeutics can be designed and implemented.
  • Figure 4 depicts another exemplary coupling chemistry in which X is Br or I.
  • Carbonyl activates the nearby halogen so that it is easily substituted by a strong nucleophile such as a negative sulfur atom.
  • the PEG has a terminal thiol group, and the other end has biotin.
  • the liposome has 1 - 5 % bromo-, or iodoacetyl cardiolipin.
  • Cardiolipin (For example, see structure below) is an exemplary phospholipid for anchoring the peptides to liposomes and other lipid-based delivery vehicles. Although cardiolipin has been used in liposomes for drug delivery, it generally is not used for anchoring purposes. Cardiolipin liposomes tend to go to the heart, because the heart has more cardiolipin than other organs. To avoid this, a small amount of cardiolipin that is chemically modified is used. Cardiolipin has one free hydroxyl group that will be is esterified with bromo-, or iodoacetyl moiety for the binding of peptides via PEG.
  • Thiol-PEG is added under slightly basic conditions to form a thioether bond.
  • Streptavidin can be bound as described above. If the PEG has a carboxyl group instead of biotin on the other end, EDC can be used to couple peptides directly with PEG. Cardiolipin has four fatty acids instead of two like all the other phospholipids. This provides stronger anchoring of the peptides to the liposome.
  • Exemplary of a delivery vehicle produced as described above and in the Examples, are liposomes displaying a plurality of different peptides including at least two proglucagon peptides and an anabolic peptide.
  • liposomes that display:
  • PeptideFLl GLP-l
  • PeptideFL4 Oxyntomodulin
  • the delivery vehicles with linked peptides can be formulated for any suitable route of delivery, including, but not limited to, injection, inhalation, mucosal, and other routes.
  • the route includes the indication treated or tissue targeted and the particular delivery vehicle.
  • Many delivery vehicles cannot be orally administered; some such as certain exosomes can be orally administered.
  • the contemplated route of administration is injection. Pens and syringes containing formulations of the delivery vehicles, as a single dose or multiple doses, are provided.
  • the delivery vehicles such as liposomes
  • the peptides are provided on delivery vehicles, such as liposomes.
  • the delivery vehicles can display at least two peptides for weight loss, and at least one for muscle enhancement, or combinations of delivery vehicles each displaying one peptide are administered together.
  • the regimens as described below administer that therapeutics displayed on or linked to or embedded in delivery vehicles.
  • the activation of cell surface receptors can trigger regulatory processes that restrict signaling duration and/or strength.
  • Downregulation is a process by which a particular cellular component, such as a protein (e.g., receptor), decreases in abundance or decreases activity responsive to an external stimulus (z.e., long term administration of an agonist).
  • a specific receptor or decrease in total receptor number in the cell can decrease (z.e., by enhanced receptor degradation or decreased receptor synthesis or ligand-induced internalization of receptors (endocytic downregulation)).
  • the decrease in receptor activation or number can reduce cell sensitivity to the agonist.
  • the receptor system In the presence of sustained ligand stimuli, the receptor system enters a refractory state thereby preventing the cell from over-responding to the ligand.
  • This negative feedback mechanism is important in drug treatment, where chronic exposure to a drug and/or therapeutic molecule can become less effective upon long-term or chronic exposure.
  • An example of this negative feedback mechanism are hormone receptors feedback systems. When receptors have been chronically exposed to an excessive amount of a ligand or exposed to a ligand for a prolonged period of time, either from endogenous mediators or from exogenous drugs, this can result in ligand-induced desensitization or internalization of that receptor. Downregulation, therefore, effectively minimizes overstimulation of a pathway to prevent disruption of an organism’s internal processes as a result of the increased or chronic activation.
  • Downregulation is a cellular mechanism characteristic of long-term administration of therapeutic agents. Understanding downregulation and cellular responses to chronic drug exposure is fundamental to developing an effective drug regimen for treating a complex disease state. Targeted therapeutics that are rotated to decrease receptor or cellular downregulation, or other cellular responses that decrease efficacy or activity of a therapeutic agent can be used to treat diseases, disorders, and conditions, such as obesity, and have wide reaching implications across medical specialties for drug development for chronic conditions in which there are a plurality of targets for therapeutic intervention for in which desensitization to treatments occurs.
  • Combinatorial treatments involve the use of two or more, generally at least three, different treatments that target different pathways for treating a disease, disorder, or condition.
  • the combinatorial treatments particularly if they start to lose effectiveness for treatment of chronic diseases, disorders, and conditions, can be rotated, which avoids the problems, particularly desensitization, which occurs with long-term therapies for diseases, disorders, and conditions, such as obesity, Parkinson’s disease, and other chronic diseases, disorders, and conditions that require treatment for many months, and generally for life.
  • Rotational combinatorial therapy involves protocols/regimens for treatment of a disease, disorder, and/or condition, in which two or more different therapies (or therapeutics) in combination are administered such that the combinations of the -n- therapies are rotated for predetermined or multiple rounds of treatment.
  • Rotational combinatorial therapy or pharmacology is a protocol for treating a disease, disorder, and/or condition, in which a number of therapeutics or treatments, at least two, different therapeutics/treatments for a disease are administered according to a schedule in which different therapeutics and combinations thereof are administered. Each therapeutic/treatment can be different, having a different target or mode of action.
  • a combination in the rotational protocol can contain more than one therapeutic, wherein each targets the same pathway to effect a therapeutic effect, which can, in some instances, achieve a synergistic effect such that lower doses, compared to monotherapy, of each therapeutic can be administered.
  • the timing for rotation can be decreased. Because the medications are used in a therapeutic combination, in some examples a medication or medications in the combination can be used at lower dosages than treatment with a monotherapy, to reduce the risk or severity of adverse side effects.
  • each combination of treatments is administered for a predetermined time, generally at least a week, and then replaced by a different combination, which is administered for a predetermined period of time.
  • Each combination can include a drug in common, and/or one of the rotations can include only a single therapeutic, but each combination is different from the other combinations.
  • a rotational protocol can include at least two different combinations that are rotated, and generally includes at least three combinations of therapeutics and treatments that are rotated. The protocol can be repeated a plurality of times.
  • the predetermined rounds for treatment and timing for treatment can be modified if the subject demonstrates signs or signals that a pathway is downregulated, or the subject becomes less responsive to the therapeutic effects of the therapy or the subject exhibits an increase in adverse side effects.
  • the combinations can be switched prior to the predetermined time.
  • the subject exhibits a decrease in the side effects of the medication this can indicate that downregulation of the pathway has occurred or is occurring, and the dosage can be increased or another therapeutic that activates a different pathway can be substituted.
  • the diminution can indicate that the therapeutic pathway is downregulated and medication that acts through a different pathway can be substituted.
  • the regimen can involve a first round in which one, two, or more of the therapeutics/treatments are administered for a period of time, followed by second round in which a different therapeutic/treatment or different combination of therapeutics is/are administered for another period of time, and repeating round one and round two, or administering a third round of pharmaceuticals for a predetermined period of time.
  • different combinations of therapeutics are administered for each time period, providing different combinations of therapeutics rotated for predetermined periods of time.
  • the time periods for treatment are not predetermined; the different combinations are administered for each period of time, and the combinations of therapeutics are rotated when the patient is showing reduced therapeutic benefit(s), or the patient is showing increased or intolerable side effects from the therapeutics.
  • the therapy involves at least two different therapeutics, administered sequentially, and then together, or administered together, and typically involves at least three different therapeutics, usually drugs, administered separately or in combinations. Generally, all combinations and orders of administration can be included in the protocol. Hence, the name rotational combinatorial pharmacology.
  • a regimen of a rotational combinatorial therapy described herein comprises two or more different therapeutic combinations (also referred to as clusters), where each combination includes two or more medications or treatments. Each combination is rotated for a predetermined time of treatment. Rotation can revert to combination one or any other combination in the regimen.
  • a rotational combinatorial treatment or therapy includes the administration of a first therapeutic agent or combination of therapeutic agents and, after a set time point and in fixed intervals, administration of a different therapeutic agent or combination (cluster) of therapeutic agents, where the second combination (cluster) of agent(s) target a receptor/and or pathway distinct from at least one of the initial therapeutic agent(s), and, optionally, any subsequent combinations are administered after a set time point and in fixed intervals.
  • the first therapeutic agent(s) combination is/are administered and then, such as three months later, a second therapeutic agent(s) combination is/are administered.
  • treatment can proceed for years, and can, if necessary be administered for life.
  • Any of the therapeutic agents used in the rotational therapy herein can be a pharmaceutical or non-pharmaceutical therapy.
  • the combinations can be administered in accord with any of the regimens set forth in the any of following tables:
  • Tables 4 and 5, below set forth rotational combinatorial therapy regimens that are rotated at 4-month and 6-month intervals, respectively.
  • downregulation generally occurs after at least 3 months and, thus, rotations of combinations generally occur at or about 3 months or later.
  • regimens such as those set forth in Tables 4 and 5 are employed.
  • Therapeutic #8 tesamorelin
  • Therapeutic #9 Pancreatic polypeptide (PP)
  • the combinatorial treatment or therapy and rotational combinatorial treatment or therapy provides an improved treatment of diseases and disorders compared to treatment with monotherapies, such as disease states that result from derangements of multiple molecular or cellular pathways or a that show a decrease or plateau in the efficacy of the monotherapy; the combinatorial therapy and/or rotational combinatorial therapy described herein modulates the multiple pathways derangements to ameliorate negative effects of the disease or disorder. Modulation of multiple pathways provides improved therapeutic benefits compared to monotherapy or even dual therapy, which target fewer molecular or cellular targets or pathways. Rotating multiple therapeutics (z.e., pharmaceuticals) is an effective method of preventing molecular and cellular adaptation that occurs after continued (z.e., longterm) treatment with monotherapy.
  • Rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with combination therapy (z.e., treatment with more than one therapeutic) that is not rotated.
  • rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with a monotherapy or combination therapy that is administered for a prolonged period (e.g., longer than 3 months).
  • the therapeutics in the combination therapy and/or rotational combinatorial therapy are synergistic; one therapeutic (z.e., drug) enhances the clinical activity of another therapeutic (z.e., drug) when used in the combination, and the cumulative effects of the therapeutics exceed the expected clinical benefit of the sum of the multiple drugs in the combination.
  • increased efficacy of the combination works through independent drug action rather than a synergistic effect of the therapeutics in combination; the therapeutic benefit is attributable to a single therapeutic in the combination and the benefits over monotherapy are due to increasing the odds that the combination includes a drug that is effective for a particular patient.
  • combination therapies for use in a rotational regimen for use in the treatment of chronic conditions, such as conditions lasting more than three months, and conditions where patients develop a tolerance to treatment.
  • the rotational combinatorial therapy is for treating chronic conditions with a plurality of known treatments or known pathways associated with disease progression or pathology.
  • a rotational combinatorial therapy regimen can be developed by identifying known treatments/therapies the disease(s), disorder(s), and/or condition(s) for treatment; identifying the pathways, mechanism of actions or targets for treatment; selecting treatment(s) and/or therapies that include treatment(s) and/or therapies that activate different pathways or that have different mechanisms of action; creating combinations that include at least two therapeutics known to activate different molecular and/or cellular pathways and design a regimen for administration of the combinations; and creating a regimen to administer multiple rounds of treatment, with different combinations.
  • combinations used in one or more rounds of rotation can include a single therapeutic.
  • all combinations that are rotated include two or more therapeutics or treatments.
  • the combinatorial rotational therapy provided herein can be used in methods of therapy for treating diseases, disorders and/or conditions where the affected patients are at risk of developing a tolerance or resistance to the therapeutic effects of the therapeutic(s) (z.e., medications).
  • the diseases, disorders, and/or conditions have more than one target/pathway for therapeutic intervention.
  • the disease, disorder, or condition is chronic or requires extended or life-long treatment.
  • Other diseases, disorders and/or conditions are those where the afflicted individuals are at risk of developing a tolerance or resistance to the therapeutic effects of the medications and include chronic conditions, where the disorder or its effects are persistent or long-lasting, or is a disease that develops over time.
  • Patients with a variety of diseases or conditions can benefit from a rotational combinatorial therapy described herein.
  • These conditions include, for example those that can be treated by: 1) a combination of more than one medication and 2) a rotational aspect, such as conditions where the prescribed therapeutics have the potential to decrease efficacy over time.
  • the methods herein address the problems of inadequate effectiveness of a monotherapy, and the desensitization. Combinations of a plurality of drugs can improve effectiveness; combining this with rotation of the combinations of the drugs (or of the drugs), leads to sustained weight loss.
  • a disease, disorder, or condition for treatment with a rotational combinatorial therapy described herein is one where 1) there are a plurality of different therapeutics/treatments known or that can be developed for treating the disease, disorder, or condition, such as where there are a plurality of therapeutic intervention pathways or targets; and/or 2) the disease, disorder, or condition generally is chronic; and/or 3) the disease, disorder, or condition is one for which treatment often fails because tolerance to the therapeutics/treatments develops and/or the therapeutics/treatments become ineffective over time.
  • the rotational combinatorial therapy provided herein can be used for treatment of any condition where there is an effective treatment or a plurality of different known therapeutics and/or treatments.
  • the rotational combinatorial therapy provided herein can be used for treatment of any condition where there is a known pharmaceutical or plurality of pharmaceuticals that ameliorate(s) symptoms of the condition.
  • the rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, such that a therapeutic can be developed for treating the disease or disorder or condition
  • a disease, disorder, or condition in which there are a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways that are involved in disease progression can be treated with the rotational combinatorial therapy described herein.
  • the rotational combinatorial therapy described herein can be used to target multiple pathways that are associated with a disease or disorder, but where a pharmacotherapy has not yet been developed.
  • activation of the receptor(s) can lead to activation of an array of cellular pathways.
  • Administration of a monotherapy, which activates or effects activation of a single pathway can have limited efficacy for improving the disorder.
  • An approach for reducing the symptoms or effects of the disorder can require administering a combination of compounds targeting the different pathways and processes.
  • the combination therapies described herein can include treatments and/or therapies that target multiple pathways, and diseases or conditions for treatment where targeting a single pathway or mechanism will not confer significant or complete amelioration of the disease state or symptoms.
  • the rotational combinatorial therapy provided herein can be used to diseases where administration of a single therapeutic in animal models or in human patients has a limited therapeutic effect.
  • the disease, disorder, and/or condition is a multifactorial process where the therapeutic benefit from a combination of different therapeutics exceeds the therapeutic benefit of a monotherapy.
  • the disease, disorder, or condition is influenced by multiple genes (polygenic) and resultant genetic pathways, and generally in combination with lifestyle and environmental factors, such as exercise, diet, or pollutant exposures. The combination of genetic and environmental factors act together in concert to trigger the development and progression of the multifactorial disease.
  • rotational and/or combinatorial pharmacology provides a method for treating complex disease processes, such as obesity, which involve multiple pathways, in which monotherapy and dual therapy have shown limited success.
  • the combinatorial pharmacology for weight loss as exemplified herein is exemplary of a condition that can be treated with a rotational therapy.
  • Other conditions that involve multiple molecular and/or cellular pathways also can be treated by rotational combinatorial pharmacology, such as conditions where monotherapies are inadequate.
  • Combination therapy also can overcome limitations of monotherapies, where multiple medications, by virtue of the number of medications in the combination, increases the chance the subject is responsive to a particular therapeutic. b. Chronic Conditions
  • Chronic conditions require ongoing therapeutic intervention and can negatively impact or limit the daily activities or the quality of life of the affected individual.
  • Chronic conditions include conditions in which known therapeutics are ineffective or that do not significantly or completely ameliorate the symptoms or characteristics of the condition.
  • Chronic conditions also include conditions where patients treated with monotherapies are not responsive to treatment or who have not improved on the monotherapy or who continue to have adverse symptoms of the condition despite the therapeutic intervention.
  • Chronic conditions also include conditions where patients treated with combination therapies (z.e., continuous and/or prolonged administration of more than one therapeutic) are not responsive to treatment or who have not improved on the combination therapies or who continue to have adverse symptoms of the condition despite the therapeutic intervention.
  • Chronic conditions are conditions that persist over time. In some examples, chronic conditions last more than 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more.
  • the rotational combinatorial therapy provided herein is for treating chronic conditions. In some examples, the rotational combinatorial therapy described herein is for treating a chronic condition, disease, or disorder in which the course of the condition, disease or disorder lasts more than about three months.
  • a chronic condition also can be characterized by the amount of time the therapeutics for treating the condition are administered.
  • chronic conditions can be conditions in which therapeutic(s) for treating the condition is/are administered for an extended period of time (ie., about 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more).
  • Patients with chronic conditions can be administered a medication or combination of medications for months or years, during which time they can develop a decreased response to the medication(s), such as decreased response due to signal downregulation or desensitization to the medication, and can suffer from side effects from prolonged use of the medication(s).
  • Subjects with chronic conditions can benefit from treatment with combinations of therapeutics that are rotated, which can increase the likelihood of a prolonged therapeutic response compared to treatment with a monotherapy or combination of therapeutics that are not rotated.
  • combination therapies for use in a rotational regimen for use in the treatment of chronic conditions are administered therapeutic treatment(s) for extended periods of time and can develop a tolerance to treatments, such that the therapeutics for treating the conditions become or are less effective.
  • combination therapies for use in a rotational regimen for use in the treatment of chronic conditions for use in the treatment of overweight or obesity, and/or for weight loss.
  • the combination therapies for use in a rotational regimen also are for use for treating or ameliorating the symptoms of comorbidities of overweight and/or obesity.
  • Exemplary of such comorbidities include diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, depression, non-alcoholic fatty liver disease, and cancer, such as but not limited to, pancreatic cancer, breast cancer, prostate cancer, gastric cancer, colon cancer, ovarian cancer, head and neck cancer and others.
  • the combination therapy and rotational regime can be modified to decrease the amount or severity of adverse side effects associated with administration of the therapeutic(s). For example, the dosage of the therapeutic(s) can be lowered, or the therapeutics or therapeutic combinations can be rotated with greater frequency.
  • Rotating combinations of therapeutics can expose patients to each of the particular therapeutics for a shorter amount of time or to a lower dosage, which can each decrease the chance of developing side effects or the severity of side effects from the particular medication.
  • the rotational combinatorial therapy provided herein can be used to minimize side effects during treatment or therapy for diseases or disorders, compared to treatment with a monotherapy or dual therapy or combination therapy with two or more therapeutics.
  • the combination therapy described herein can be used and/or administered at a lower dosage than the dosage of the therapeutic used for monotherapy.
  • the combination therapy is used at a lower dosage than the monotherapy which results in fewer or less severe adverse side effects during treatment.
  • Patients with a chronic condition who previously were treated with a monotherapy, where the monotherapy did not decrease disease signs or symptoms or where the monotherapy did not inhibit or stop disease progression can be administered a combination therapy or rotational combinatorial therapy provided herein to improve the therapeutic response or to decrease the adverse symptoms associated with the disease state.
  • patients with a chronic condition who were previously treated with a combination therapy, where the combination therapy did not decrease disease signs or symptoms or where the combination therapy did not inhibit or stop disease progression can be administered a rotational combinatorial therapy provided herein.
  • a rotational combinatorial therapy also can be used for treating subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy and who are responsive to the treatment, but where the treatment did not completely ameliorate symptoms of the condition or did not cure the disease.
  • a patient can benefit from a combination therapy targeting multiple molecular and/or cellular to target pathways to which the subject is more responsive.
  • a combination of therapeutics administered in a rotation or regimen described herein can show improved amelioration of symptoms of a chronic condition compared to a monotherapy or combination of therapeutics that are not rotated by decreasing downregulation of therapeutic pathways and activating molecular pathways that were not previously activated (z.e., not previously subject to downregulation).
  • Patients with chronic conditions can develop a decreased response (tolerance) to the medication(s) prescribed for treating the condition or for ameliorating symptoms of the condition.
  • Such decreased therapeutic response over time can be related or due to signal downregulation or desensitization to the medication(s).
  • Patients that develop a tolerance to a monotherapy or combination therapy can benefit from a rotational combinatorial therapy to overcome the desensitization, such as desensitization due to receptor downregulation.
  • a rotational combinatorial therapy thus, can be used for treating conditions where patients develop a tolerance to treatments.
  • subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy who are responsive to the treatment z.e., showed an amelioration of symptoms
  • subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy who are responsive to the treatment z.e., showed an amelioration of symptoms
  • the treatment was less effective or the subject failed to respond to the treatment after a time period
  • a combinatorial rotational therapy can be treated with a combinatorial rotational therapy.
  • a subject who initially showed amelioration of symptoms of the disease or condition, but where the symptoms increased after prolonged treatment such as, for example, a subject who showed amelioration of symptoms but where symptoms increased after at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment.
  • Rotational combinatorial therapies provided herein are for treating a disease, disorder, and/or conditions where there are a plurality of different therapeutics/treatments known or that can be developed for treating the disease, disorder, or condition, and the disease, disorder, or condition generally is chronic and/or is one for which treatment fails because tolerance to the therapeutics/treatments develops or the therapeutics/treatments become ineffective over time so that ultimately treatment of the disease, disorder, or condition fails or becomes increasingly ineffective.
  • development of a rotational combinatorial therapy can include a protocol including:
  • a protocol for developing a rotational combination therapy can include identification of treatments or therapies that are known and/or have previously been characterized for treating a disease, disorder, or condition.
  • a protocol for developing a rotational combination therapy described herein can include identifying treatments or therapeutics that have previously demonstrated activity or efficacy for treating the disease, disorder, or condition or for treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition.
  • a therapy or therapeutic is a drug that is approved by the Food and Drug Administration (FDA) for human use in the United States.
  • the therapeutic can show efficacy for treating the disease, disorder, or condition or treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition in animal models.
  • the therapeutic (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that was previously identified as a therapeutic that alters (z.e., activates or inhibits) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and/or ex vivo model systems.
  • the in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug/pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is/are implicated or use of a particular therapeutic is predicted to be effective.
  • a therapy or therapies is/are effective for treating or ameliorating a disease, disorder, or condition can be used to select therapeutics for inclusion in the rotational combination therapy described herein.
  • the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be identified in screens for effective therapeutics for treating a disease or disorder. For example, in vitro screens in model systems that replicate disease characteristics can be conducted for new substances showing activity and additionally for assessing effectiveness for treating a disorder.
  • the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein are identified in vitro in screens conducted in cell culture, in a Boyden chamber, in three-dimensional cultures, in microfluidic systems, using 3D bioprinting, or in other systems that can be used to identify therapeutics that can be for treating a disease, disorder, or condition with the rotational combinatorial therapy described herein.
  • known weight loss medications such as weight loss medications described herein can be included in the combination.
  • a previously identified weight loss medication can be included, including one or more of mitochondrial uncouplers, amphetamines, thyroid hormones, drug cocktails, neuromodulators, lipase inhibitors, cannabinoid receptor antagonists, gastrointestinal-derived peptides chemically optimized for pharmaceutical use, and others.
  • one or more FDA approved medications can be included in the combination therapy and/or incorporated into a liposome, generally into a liposome that displays one or more of the peptides.
  • one or more of orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, tirzepatide (is a GIP analog that acts on GIP and GLP-1 receptors; sold under the trademark Mounjaro®) dual agonist, and semaglutide can be included in the combinations described herein for treating obesity or overweight or for effecting weight loss.
  • FDA approved medications that are for use for a disease or disorder where the subjects exhibit weight loss as a result of the therapeutic regimen, but for which weight loss is not the primary objective, can be included in the combination therapy provided herein, for effecting weight loss.
  • a protocol for developing a rotational combinatorial therapy can also include identification of pathways, such as, for example, molecular or cellular pathways, associated with the disease, disorder, or condition.
  • the molecular and/or cellular pathways can be used to determine the therapeutic(s) for inclusion in the combinatorial therapy.
  • Treatments or therapeutics for inclusion in the rotational combinatorial therapy described herein can be selected from compounds that target a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition. Treatments or therapeutics can activate a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that activation of the pathway ameliorates symptoms of the disease, disorder, or condition or secondary side effects.
  • treatments or therapeutics can inhibit a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that inhibition of the pathway (z.e., inhibition of an overactive pathway or an off-target pathway) ameliorates symptoms of the disease, disorder, or condition or secondary side effects.
  • biomarkers can signal an abnormal process or a condition of the disease.
  • Biomarkers can be used to identify targets for treatment and potential responses to therapeutics and therapeutic combinations.
  • biomarkers can be used to predict a patient’s response to individual therapeutics (z.e., drugs) in a combination.
  • higher receptor expression in a patient can correspond to a better response to a corresponding therapy, and lower expression of receptor activators can indicate a higher or lower chance of responsiveness to a particular therapeutic(s) (z.e., drugs).
  • biomarker identification and characterization, and use of validated biomarkers can help predict patient response and therapy -related side effects and inform selection of therapeutics for inclusion in the combination and/or rotational combinatorial therapy herein.
  • the skilled artisan can evaluate the literature, for example articles published in the scientific literature, such as, for example, in the US National Library of Medicine and the National Center for Biotechnology Information, which assembles biomedical literature from MEDLINE, life science journals, and online books.
  • the scientific literature can identify and assess molecular and cellular pathways for treating diseases, disorders, and/or conditions described herein.
  • Molecular targets for treating a disease, disorder, or condition can be identified and/or evaluated in the scientific literature and used to determine molecular targets for therapeutics for treating the conditions identified and described herein.
  • a pathway associated with the disease, disorder, or condition or symptoms thereof for treatment with a combination or rotational combinatorial therapy described herein can be identified using in vitro, in vivo, and/or ex vivo model systems.
  • the in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug/pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is implicated or use of a particular therapeutic is predicted to be effective.
  • a molecular and/or cellular pathway plays a role in pathogenesis or progression of a disease or condition can be used to determine therapeutics for including in the combination and rotational combination therapy described herein.
  • the therapeutic (i.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that alters (i.e., activate or inhibit) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and/or ex vivo model systems.
  • molecular and/or cellular pathways that are altered in gastric bypass patients can be targeted in the therapy by combining therapeutics that target each pathway.
  • molecular and/or cellular pathways that are altered following gastric bypass or other surgical weight loss procedures can be used to formulate a pharmacotherapy for inclusion in the combination and/or rotational combination therapy described herein.
  • peptides that mimic gut peptides or that are gut peptide receptor agonists; or therapeutics that alter expression and/or activation and/or activity of neuro-hormonal gut peptides that are altered following bariatric surgery, such as, for example, therapeutics that increase expression and/or activation and/or activity of neuro-hormonal gut peptides that are increased following bariatric surgery, can be included in the combination therapy described herein.
  • the combinatorial regimens and the rotational combinatorial regimens can mimic the effects of bariatric surgery, particularly gastric bypass surgery, such as the Roux-en-Y surgery.
  • Gastric bypass surgery has effects that include decreased absorption of food, including intestinal absorption, on glucose homeostasis, and results in hormonal changes that alter appetite/satiety and energy consumption and other physiological processes.
  • Rerouting food through the gastrointestinal tract leads to changes in gut hormone secretion.
  • Changes in gut hormone levels after RYGB include increased anorectic hormones, such as GLP-1 and PYY, which induce satiety, and decreased levels of orexigens, such as ghrelin, an appetite-stimulating hormone.
  • the rotational combinatorial methods herein allow a variety of combinations of drugs/treatment to reduce desensitization, and adverse effects, while providing the advantages of gastric bypass surgery by combining administration of various drugs that reduce appetite, and hormones that alter appetite. Combinations of such drugs and treatments are detailed herein and are exemplified in the working examples and throughout the description.
  • combinatorial methods and combinatorial rotational methods in which combinations of medications, such as peptide hormones or agonists thereof and/or antagonists of peptide hormones that are reduced following bariatric surgery, are administered. Particular combinations are described herein and exemplified in the working examples.
  • molecular and/or cellular pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and or therapeutics for the combination therapy.
  • Combination therapies containing at least two, and generally at least three, combinations of a plurality of therapeutics and rotated to prevent desensitization of activated pathways exhibit efficacy that is greater than a monotherapy or combination therapy that is not rotated.
  • the combinations contain therapeutics that activate or are known to activate different molecular or cellular pathways.
  • the combinations contain therapeutics that activate or are known to activate a total of at least two, three, four, five or more different molecular or cellular pathways.
  • Therapeutics that decrease in efficacy over time can be included in the combinations herein, including, for example, pharmaceutical medications that activate molecular pathways that are downregulated, leading to decreased efficacy of the pharmaceutical.
  • the combinatorial therapy herein includes rotating from one combination comprising a plurality of therapeutics to a next combination comprising a plurality of therapeutics.
  • the rotational aspect overcomes limitations associated with therapeutics that decrease in efficacy over time; the switch to a new combination results in a continuous therapeutic effect that does not wane, or wanes less than treatment with a monotherapy containing one of the therapeutics in the combination.
  • therapeutics for inclusion in the combination for the rotational combinatorial therapy herein are those that do not have severe adverse side effects upon ceasing use of the particular therapeutic. For example, therapeutics that do not induce withdrawal symptoms as a result of ceasing or reducing use of the therapeutic (z.e., medication). When selecting medications for inclusion in the combinations, medications that induce withdrawal syndromes and rebound upon discontinuation can be avoided. Therapeutics for inclusion in the rotational combinatorial therapy are those with a low chance of withdrawal or adverse events upon switching to the next combination of medications or those that do not induce adverse events upon switching to the next combination of medications.
  • therapeutics for inclusion in the combinatorial therapy are those that are not delayed in producing a therapeutic effect or are not delayed in ameliorating symptoms of the disease or condition to be treated.
  • the therapeutics included in the rotational combinatorial therapy are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or more, and in some examples rotated at least every 3 months.
  • the therapeutic(s) takes effect (z.e., has activity) or the therapeutic effect(s) is/are produced or amelioration of symptoms begins near to the time of initial administration, such as within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment.
  • the therapeutic(s) takes effect (ie., has activity) or the therapeutic effect(s) is/are produced or amelioration of symptoms begins within at least 1 week of initial administration.
  • the therapeutic activity can begin within the first half, third, quarter, or tenth of the rotational time period.
  • the therapeutics in the combinations can show therapeutic activity within 1 month, and more generally within at least 2 weeks or 1 week or less than 1 week, such as two or three days after initial administration.
  • Medications that are toxic if administered at high dosages can be included in the rotational combinatorial therapy described herein.
  • Therapeutics ie., drugs
  • the therapeutics used in combination with other, complementary, therapeutics can be administered in lower dosages such that when they are administered in combination with other therapeutics, and in a rotational regimen as described herein, they effect amelioration of symptoms or treatment of the disease, disorder, or condition without the adverse effects.
  • Administration of therapeutics in a rotational regimen described herein can reduce drug toxicity, and adverse side effects, while maintaining or improving clinical efficacy.
  • a particular medication in the combination is administered at a lower dosage than the medication would be administered in a monotherapy; administration of the medication at the lower dosage decreases drug associated toxicity and adverse side effects.
  • Previous treatment can be used to predict a patient’s response to a therapeutic or class of therapeutics or to similar therapeutics (ie., therapeutics that act through the same molecular pathway).
  • Previous treatment also can be used to predict whether targeting a particular molecular pathway will be effective in that patient.
  • Patients who have shown a positive response to therapeutics targeting one molecular pathway can be treated with a rotational combinatorial therapy containing therapeutics that target that same pathway.
  • Information from patients who were not previously responsive, or did not show amelioration of symptoms, after treatment with a monotherapy targeting one molecular pathway, can be used to determine the therapeutics for inclusion in a combination therapy herein.
  • a plurality of pharmaceuticals targeting the molecular pathway can be included in a combination therapy.
  • the pharmaceutical can be excluded from the combination therapy.
  • the pharmaceutical can be included in the combination therapy with other therapeutics predicted to act synergistically with the pharmaceutical.
  • Treatments that target different pathways associated with satiation, metabolism, hunger and weight gain or loss can be included in a rotational combinatorial therapy herein for treating overweight, obesity or for weight loss, or for treating comorbidities of overweight or obesity, such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (z.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, cancer, depression, and non-alcoholic fatty liver disease.
  • therapeutics that mimic the physiological effects following gastric bypass can include in a rotational combinatorial regimen as provided herein.
  • the combination can include therapeutics that target the same pathways or that target different pathways.
  • Therapeutics that can provide complementary benefits (z.e., modulate similar or the same molecular pathways to compound the effects), or therapeutics that act through different molecular pathways to effect positive benefits through a variety of pathways can each be included in the rotational combinatorial therapy described herein.
  • the therapeutics in the combinations generally target different pathways. Selecting therapeutics that target different pathways is generally to prevent redundancy in the pathways that the therapeutics target, such as, for example, where redundancy does not increase the therapeutic benefit over the benefit of a single therapeutic, or when redundancy can increase the likelihood or severity of adverse side effects.
  • Selecting therapeutics that target different pathways also overcomes the decreased therapeutic effect that occurs after desensitization of a particular molecular pathway after prolonged treatment. For example, if a therapeutic in a first combination targets a pathway that is desensitized after use, the therapeutic in the second combination targets a second pathway, which is not desensitized, and, thus, not downregulated; a third combination can contain the first therapeutic, if the desensitization no longer exists, or can contain a third therapeutic that targets a third pathway. The rotation of therapeutics that target different pathways overcomes the reduced therapeutic effect after desensitization of a pathway.
  • the therapeutics for use in the rotational combinatorial therapy described herein can be therapeutics that are complementary in that one effects treatment of secondary effects (z.e., inflammation) and the other effects treatment of the primary cause of the disorder.
  • one therapeutic in the combination can inhibit production of proinflammatory cytokines that are a downstream effect of the primary disorder, such as, cancer and obesity.
  • the combination can include therapies that treat or ameliorate the symptoms of concomitant disease states, such as a treatment (z.e., medication) for hypertension included in a combination for treatment of the primary condition of obesity.
  • Therapeutics that can provide complementary benefits by effecting treatment of different disease phases can be selected for inclusion in the rotational combinatorial therapy described herein.
  • one therapeutic or combination of therapeutics can effect treatment or amelioration of effects of an acute phase of the disease and the other therapeutic(s) can effect treatment or amelioration of effects of a chronic phase or state of the disease.
  • the overlap is, for example, to ensure the efficacy of the combination, to ensure activation or inhibition of a particular pathway.
  • Therapeutics with overlapping targets also can be included in separate combinations in the therapeutic regimen, such as, for example, combinations that are administered at different points in the therapeutic regimen, and generally with a cycle of the regimen separating administration of the redundant therapeutics.
  • the combinatorial rotational therapy described herein includes a regimen for administering the plurality of combinations multiple times, such that there are at least two rounds, and generally at least three, four, five or more rounds of treatment, with different combinations.
  • Various factors can influence the timing for administering the plurality of combinations in the rotational combinatorial therapy provided herein including, but not limited to: the particular therapeutics in the combinations; the particular disease to be treated; the predicted and/or known efficacy of the therapeutics or therapies, alone or in combination with other therapeutics and/or therapies; the time for the therapeutics to demonstrate therapeutic activity (e.g., onset of action); the recommended time for therapeutic administration; the maximum recommended time for therapeutic administration; medication dosages; the characteristics of a patient to be treated, including, but not limited to, gender, weight, age, overall health, comorbidities, and other characteristics; and other particulars of the therapeutic(s), patient and disease, disorder, or condition.
  • the first combination in the rotational combinatorial therapy can include a therapeutic or therapeutics with a high (z.e., the highest) probability of activity and/or efficacy for treating the disease, disorder, or condition.
  • a therapeutic or therapeutics with a high (z.e., the highest) probability of activity and/or efficacy for treating the disease, disorder, or condition.
  • lower dosages or a less aggressive first combination of therapeutics can be administered.
  • the therapeutics for inclusion in the first line therapy can depend on the disease, disorder, or condition, to be treated, the disease state, state, the rate of progression of the disease, disorder, or condition, the severity of the disease, disease, disorder, or conditions.
  • the skilled physician can modify the therapeutics for inclusion in the rotational combinatorial therapy described herein based on the available therapeutics, and, for example, the disease to be treated, the disease state, the rate of disease progression, the severity of the disease, the particular patient to be treated, and other factors.
  • the rotational aspect of the combination therapy provided herein provides for changing of doses and replacement of ineffective or less effective therapeutics with others that have increase efficacy, for example, based on the patient response.
  • the time for the drug or therapeutic to exhibit therapeutic activity also can be considered when selecting a drug or other therapeutic for treatment in a rotational treatment regimen provided herein.
  • a drug or therapeutic used in the rotational combination therapy herein should be active prior to rotation and administration of the next therapeutic combination. For example, if a medication takes one week to show therapeutic activity, the combinations will not be rotated at less than one week. For example, if a medication takes one week to show therapeutic activity, the combinations can be rotated after at least one month, two months, three months or more, in order to show therapeutic activity of the initial combination.
  • the timing for rotation of the therapeutic combinations can be determined.
  • Various factors can be considered to identify indicators for a switch from one therapy (z.e., treatment or drug or therapeutic or combination thereof) to another, and the timing for a switch.
  • the following factors can influence the decision to switch medications: one or more adverse side effects; change in clinical status (e.g., improvement or worsening of the disorder or condition to be treated); poor drug activity despite administration of a therapeutically effective amount of drug; concomitant pharmacotherapy (i.e., drug-drug interactions); need for a different route of administration (e.g., oral vs.
  • combinations are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or 9 months or 1 year or 15 months or 18 months or 2 years or 27 months or 30 months or 33 months or 4 years or more, and in some examples rotated at least every 3 months.
  • downregulation can occur after at or about 3 months or less, the combinations can be rotated every three months.
  • the regimen can be modified to improve the therapeutic effects.
  • the patient’s clinical status can be used to modify the treatment regimen or schedule or drugs administered. For example, if a patient develops drug sensitivities, the regimen can be modified to switch to a new therapeutic under an expedited schedule.
  • the rotational schedule can be extended if previously unrealized benefits of the therapeutic are achieved with a particular medication.
  • the dosage of the individual therapeutics in the combinations for rotation can be determined. Various factors can be considered to identify the dosage for each therapeutic (ie., drug).
  • the examples and disclosure provided herein can be used to develop guidelines for a rotation schedule, and identify the medications for inclusion in the regimen/schedule.
  • the dose of an individual therapeutic can vary depending on the other therapeutics in the combination, and can vary depending on the doses of the therapeutics in the other combinations in the rotation.
  • the regimen can include guidelines for dose adjustment based on the action of the previously administered medications, relative potency, and other facts that can influence efficacy of the therapeutic(s).
  • the dose range for each of the individual therapeutics in the combinations provided herein can be adjusted by monitoring the subjects (z.e., patients), such as monitoring plasms or monitoring symptoms or monitoring adverse side effects after administration of the combination(s).
  • the dose administered can be such that the subject will maintain a plasma level to effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered.
  • the dose also can be a dosage that will effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered where the subject does not exhibit adverse side effects, or does not exhibit adverse side effects at a severity that will effect cessation of the treatment.
  • the combinatorial and rotational combinatorial therapy provided herein can be used in methods of therapy for treating chronic conditions, such as, for example, overweight and obesity.
  • the combinatorial and rotational combinatorial therapy described herein can be used in methods of therapy for treating chronic conditions such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and non-alcoholic fatty liver disease.
  • a combination therapy is administered in a rotational regimen to a subject having a chronic condition.
  • the first combination of therapeutics, in a combination regimen with a second (and third or more) combination(s) far exceeds the effects of a monotherapy or combination therapy that is not rotated, for treating a chronic condition, such as, for example, obesity.
  • the effects can be synergistic.
  • extent and level of weight loss observed following the combination therapy provided herein achieves results that have heretofore not been achieved with existing non-surgical weight loss therapies, including increased fat loss and muscle gain that surpasses existing treatment regimens.
  • Combinatorial and pulsed-dose pharmacology have been used for a variety of diseases, disorders, and conditions, such as pain control, birth control and treatment of cancer, COPD, Parkinson’s disease, Alzheimer’s disease, and bacterial infections. These previous uses do not contemplate administering a combination of at least two, generally, at least three, different therapeutics/treatments (i.e., therapeutics having a different target or different mode of action) according to a schedule in which different therapeutics are rotated, and/or the combinations selected are not selected to target different pathways.
  • Treatment strategies to deal with complex medical issues such as cancer, pain control and pulmonary issues also are described herein to integrate more than one medication (combinatorial pharmacology) administered in a rotational regimen, to improve clinical outcomes.
  • the methods and regimens provided herein are designed to target different pathways involved in disease, disorder, or condition, and to rotate combinations of treatments.
  • rotational combinatorial therapy for treating complex medical disorders include first identifying known treatments/therapies for each disease state or pathways, and mechanisms of action or targets associated with the disease. Then, treatments/therapies that activate different pathways, have different mechanism of actions or targets, and/or that are compatible with a rotational therapy can be identified. Using previous treatments and pathways/mechanisms/targets associated with the disease state, combinations that include at least 2 therapeutics known to activate different molecular and/or cellular pathways can be selected and a regimen to administer multiple rounds of treatment, with combinations of therapeutics, can be designed. As set forth herein, the methods for selecting therapeutics for inclusion in a rotational combinatorial regimen, and the timing for administration of the therapeutic combinations can be prepared. In examples, the combinations can be administered in rotational regimens for treating, for example, any of the diseases, disorders and/or conditions set forth below. a. Combination Therapies in Cancer
  • ABVD Advanced Biharmonic Deformation
  • Bleomycin Vinblastine dacarbazine can be used as the initial chemotherapy treatment for newly diagnosed Hodgkin lymphoma.
  • ABVD has been the most effective and least toxic chemotherapy regimen available for treating early-stage Hodgkin Lymphoma.
  • One cycle of ABVD chemotherapy is typically given over 4 weeks in two doses, where the first dose of the drug combination is administered on day 1 and the second dose is administered on day 15, where all four of the chemotherapy drugs (Adriamycin, Bleomycin Vinblastine dacarbazine) are administered intravenously.
  • CHOP Another combination therapy for cancer treatment is known by the acronym CHOP, which includes a combination of cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone for the treatment of non-Hodgkin lymphoma.
  • Cyclophosphamide, methotrexate, and fluorouracil (5FU), also known as CMF also are used in combination for treatment of breast cancer.
  • a chemotherapy regimen used in the clinical setting
  • a chemotherapy regimen doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin, etoposide, and prednisone
  • the BEACOPP regimen which includes administering a combination of bleomycin, etoposide, doxorubicin hydrochloride (Adriamycin), cyclophosphamide, vincristine (Oncovin), and procarbazine for treatment of Hodgkin lymphoma.
  • the BEACOPP treatment generally includes four to eight 14- or 21 -day cycles with no drugs given on days 15-21.
  • a course of BEACOPP therapy also can be combined with ABVD.
  • An exemplary BEACOPP therapy protocol is set forth in Table 7, below:
  • Opioid rotation is a strategy implemented to increase efficacy of the combination of opiates; one opiate is initially administered and when efficacy begins to wane, the first opiate is replaced by another to optimize clinical outcomes and lessen side effects.
  • This drug “exchange” substitutes one opioid for another where both medications work through a similar pathway, both targeting the mu receptor; the medications are “exchanged” rather than rotated.
  • opioids are substituted when efficiency begins to wane, not at predetermined intervals, and based on a “shared decision-making approach” between the treating physician and the patient (Fine el al.. J Pain Symptom Manage (2009) 38(3): 418-425). There is no fixed period at which opioids are exchanged, the exchange is based on clinical efficacy which may or may not correlate with the biological process of receptor downregulation.
  • rotational combinatorial therapies described herein exchanges a group of medications for a second group of medications, where the second group targets different pathways or targets than the first group.
  • opioid rotation the rotated opioids are the same, and generally is one drug, not a combination of drugs, exchanged for a second drug.
  • the rotational therapy described herein includes a regularly defined period for the rotation to occur, including regularly defined intervals, to avoid receptor downregulation and to target different receptors.
  • Treatment with oral contraceptives employs both combinatorial and pulse- dosed pharmacology treatment strategies; treatment with oral contraceptives is not rotational, the different medications are not rotated.
  • Oral contraceptives come in a variety of formulations; some formulations contain both estrogen analogs and progestin, and some contain only progestin; some pills are monophasic, delivering the same dose of hormones each day, and others are multiphasic, where the doses vary each day. Doses of the component hormones also varies among commercial oral contraceptive products. The monophasic pills employ a strategy that targets the same, one pathway for birth control each month, with a pulse of hormones, a “pulse-based strategy.”
  • Multiphasic birth control pills have both and estrogen analogs and progestin components with a fixed increasing dose that is given for set periods of time for a set period (z.e., biphasic dosing includes tablets of one strength for 7 to 10 days, then tablets of a second strength for the next 11 to 14 days; triphasic dosing includes tablets of one strength for 5 to 7 days, then tablets of a second strength for the next 5 to 9 days, and then tablets of a third strength for the next 5 to 10 days).
  • the birth control pills target the same estrogen and progesterone receptors.
  • birth control pills can contain a combination of two medications and multiphasic birth control pills can increase in dosage over the month.
  • Activation of the same set of receptors differs from the rotational combinational therapy described herein because treatment with birth control pills does not contain a rotational element where different combinations of therapeutics (z.e., medications) are administered and rotated to prevent decreased efficacy due to downregulation of receptor activity; each therapeutic in the rotational combinatorial therapy described herein has a different target or mode of action, unlike birth control pills which target the same receptors each month.
  • Present combination therapies used for treatment of cancer or for pain management or for contraceptive purposes differ from the rotational combinatorial therapy described herein, and can be modified in accord with the rotational combinatorial regimen described herein.
  • efficacy of chemotherapeutic combinations can be improved by applying the rotational combinatorial regimen described herein; chemotherapeutic combinations can be rotated in accord with a specified schedule to target different receptors or pathways to prevent receptor downregulation or decreased efficacy or increased toxicity, such as adverse side effects, which occurs after prolonged continuous use of the single combination.
  • Combination Therapies to Treat Pathogens can be improved by applying the rotational combinatorial regimen described herein; chemotherapeutic combinations can be rotated in accord with a specified schedule to target different receptors or pathways to prevent receptor downregulation or decreased efficacy or increased toxicity, such as adverse side effects, which occurs after prolonged continuous use of the single combination.
  • Combination therapies are strategies for treatment of bacterial infections to overcome or prevent drug resistance, broaden the antimicrobial spectrum, improve the efficacy, treat multi-drug resistant bacteria, and lower the dose of the individual drugs to reduce the side effects.
  • Combination therapy with multiple antibiotics have been described (Drusano et al. (2014) PLos ONE 9 7):el01311; U.S. Patent Publication No. 2021/0236589).
  • US Patent Publication No. 2021/0236589 describes selecting combinations of three antibiotics and rotating among the three different-member combinations to eliminate the possibility of developing resistance.
  • the combinations of such antibiotics can be administered in a rotational combinatorial regimen to ameliorate side effects from the particular antibiotics and to avoid development of antibiotic resistance.
  • Combination Therapies to Treat Alzheimer’s Disease can be administered in a rotational combinatorial regimen to ameliorate side effects from the particular antibiotics and to avoid development of antibiotic resistance.
  • AD Alzheimer’s disease
  • cholinesterase inhibitors such as, but not limited to, medications sold under the trademarks Aricept®, Exelon®, and Razadyne®
  • glutamate regulators such as, but not limited to the medication sold under the name Namenda®
  • Non-cognitive symptoms such as behavioral and psychological symptoms, can be treated by orexin receptor agonists (such as the medication sold under the trademark Belsomra®).
  • McLarnon 2019
  • Current Alzheimer Research 16: 1007-1017 The anti-inflammatory combination therapy described by McLarnon does not contemplate rotation of the combinations, and includes administration of a cocktail of compounds to modulate inflammatory pathways activated by microglia in response to the proinflammatory AD brain microenvironment.
  • McLarnon does not consider combination therapy for treatment of aspects of AD pathology that are not related to the chronic inflammation, such as pathology that is related to amyloid deposition or other factors, such as amyloid beta activation or altered cell signaling.
  • Table 8 Exemplary Medications for Alzheimer’s Treatment
  • these rotational combinatorial treatments for Alzheimer’s disease can include the weight loss regimens or weight loss drugs.
  • glucagon-like peptide 1 (GLP-1) receptor agonists including those described herein, exhibit neurotrophic and neuroprotective effects in amyloid-P (AP) toxicity models of Alzheimer’s disease (AD).
  • AP amyloid-P
  • AD Alzheimer’s disease
  • Combination therapies are not generally used to treat hypertension because of concerns about lowering blood pressure to unsafe levels and increased risk of adverse side effects. Generally, if medications are used in combination to treat hypertension, a first medication is administered and a second can be added to the treatment if the first medication is ineffective and/or there are no significant adverse side effects. There are combination therapies have previously been shown to be superior to monotherapy for treatment of hypertension. A combination of losartan at 50-100 mg with hydrochlorothiazide at 12.5 -25 mg lowered systolic blood pressure significantly (p ⁇ 0.001) more than either drug alone (MacDonald et al.. J of the American Heart Association DOI: 10.1161/JAHA.117.006986 (2017)).
  • Combinations of medications for the treatment of hypertension generally include two antihypertensive agents with different mechanisms of action that demonstrate enhanced efficacy compared to either medication alone.
  • the medications can be used at lower dosages than treatment with individual medications, to reduce the risk or severity of adverse side effects.
  • Combinations of drugs used for treatment of hypertension are set forth in Table 9, below:
  • combination therapies that have been employed for treatment of hypertension, including those described above, but not rotational of combinations.
  • the medications can be administered initially as a combination of two drugs, or therapy can begin with one medication and one or more medications is added to the monotherapy.
  • Any of the previously described combinations of medications for treatment of hypertension or medications known to treat the molecular and cellular pathways associated with hypertension can be modified in accord with the rotational combinatorial therapy and regimens as described herein.
  • the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications (ie., ACE inhibitors and/or diuretics).
  • Parkinson’s Disease is a progressive neurodegenerative disorder characterized by altered body movements, including tremor, stiffness, slowed movement (bradykinesia), loss of automatic or autonomic movements, and speech changes.
  • PD is not curable; a number of medical treatments are used in the management or improvement of PD symptoms, including levodopa alone or in combination with carbidopa, dopamine agonists, MAO B inhibitors (z.e., selegiline (sold under the trademark Zelapar®), rasagiline (sold under the trademark Azilect®) and safinamide (sold under the trademark Xadago®), Catechol O-methyltransferase (COMT) inhibitors.
  • MAO B inhibitors z.e., selegiline (sold under the trademark Zelapar®), rasagiline (sold under the trademark Azilect®) and safinamide (sold under the trademark Xadago®
  • Entacapone (sold under the trademark Comtan®) and opicapone (sold under the trademark Ongentys®), Anticholinergics, such as, but not limited to benztropine (sold under the trademark Cogentin®), trihexyphenidyl, and amantadine.
  • Comtan® Entacapone
  • Ongentys® opicapone
  • Anticholinergics such as, but not limited to benztropine (sold under the trademark Cogentin®), trihexyphenidyl, and amantadine.
  • any of the previously described combinations of medications for treatment of PD can be administered in accord with the rotational combinatorial therapy described herein.
  • molecular and cellular pathways associated with PD progression and/or symptoms of PD can be targeted with therapeutics for inclusion in the rotational combinatorial therapy herein.
  • therapeutics that target dopaminergic pathways or neurons or neurotransmitters or receptors can be included in the rotational combination therapy.
  • the protocols can be adjusted to administer fewer medications in combination, and rotate the combinations of therapeutics.
  • COPD Chronic Obstructive Pulmonary Disease
  • COPD chronic obstructive pulmonary disease
  • SABAs short-acting beta? agonists
  • LAMAs long-acting muscarinic antagonists
  • a triple fixed-dose combination of extra fine beclomethasone dipropionate (100 pg/puff), formoterol fumarate (6 pg/puff), and glycopyrronium bromide (12.5 pg/puff) administered via a hydrofluoroalkane pressurized metered dose inhaler is superior to fixed ICS/LABA combined therapy and also superior to the LAMA tiotropium for improving lung function and exacerbation prevention in COPD patients at risk of exacerbation.
  • combination therapies used for treatment of diseases, disorders, and conditions, including those described above. Ultimately, this combination will not avoid the problems associated with desensitization to long-term treatment.
  • These combination therapies can be improved by rotating combinations of the drugs. Any of the previously described combination therapies, where there are different targets for therapeutic intervention or can be modified by rotating combinations of therapeutics or treatments to produce a rotational combinatorial therapy regimen described herein. For example, to prevent receptor downregulation upon continuous administration or adverse side effects associated with administration of multiple medications on one day, the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications. i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions
  • Rotational combinatorial therapy such as rotational combinatorial pharmacology provided herein can be used to treat diseases, disorders and conditions that are comorbid with overweight and obesity, including, but not limited to, metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
  • metabolic syndrome e.g., obstructive sleep apnea
  • non-alcoholic fatty liver disease e.g., diabetes mellitus type 2
  • cardiovascular disease e.g., heart attack, stroke
  • elevated blood pressure e.g., elevated blood cholesterol, and elevated triglyceride levels, and others.
  • Rotational combinatorial therapy can be used to treat cardiovascular disease.
  • Rotational combinatorial therapy can be used in the treatment of cardiovascular diseases including ischemia reperfusion injury resulting from stroke, myocardial infarction, cardiopulmonary bypass, coronary artery bypass graft, angioplasty, or hemodialysis.
  • Rotational combinatorial therapy also can be used in the treatment of the inflammatory response associated with cardiopulmonary bypass that can contribute to tissue injury.
  • a combination of therapeutics can be administered in accord with a regimen described herein prior to, or in order to prevent cardiovascular disease or an adverse cardiac event.
  • a combination(s) of therapeutics can be administered in accord with a regimen described herein subsequent to a cardiac event to prevent further injury from the cardiac event or to aid in recovery from the adverse cardiac event (e.g., heart attack or stroke).
  • a combination(s) of therapeutics can be administered to a subject in accord with a regimen described herein in order to ameliorate the symptoms of cardiovascular disease or the secondary damage or effects resulting from cardiovascular disease. Amelioration of symptoms can be assessed by methods described herein or those known to the skilled physician. j. Combination Therapies to Treat Overweight and Obesity
  • Fat can be stored subcutaneously or as viscerally subcutaneous and visceral adipose tissue.
  • Subcutaneous fat lies underneath the skin and is visible, and is the fat that changes after body composition improves (i.e., fat decrease) with cardiovascular resistance training.
  • Visceral adipose tissue cannot always be seen directly and distributes around the organs, making it more dangerous to health and is more strongly associated with metabolic syndrome and diabetes compared to subcutaneous fat.
  • Visceral fat actively contributes to health because it produces cytokines and immunoregulatory hormones. Excess of cytokines and immunoregulatory hormones are seen with obesity, causing inflammation and increased risk of cardiovascular disease, immune-dysregulation and a negative effect on cells sensitivity to insulin further contributing to diabetes.
  • Obesity can induce a series of chronic metabolic diseases, such as diabetes, dyslipidemia, hypertension and nonalcoholic fatty liver disease.
  • an improved treatment for overweight or obesity can decrease risk for obesity-related comorbidities including metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), cancer, elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
  • metabolic syndrome e.g., obesity-related comorbidities
  • obstructive sleep apnea e.g., non-alcoholic fatty liver disease
  • diabetes e.g., diabetes mellitus type 2
  • cardiovascular disease e.g., heart attack, stroke
  • cancer e.g., elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
  • weight loss therapies Significant and harmful side effects are associated with weight loss therapies.
  • pharmaceutical weight loss treatments are not as effective as surgical interventions (ie., generally do not show high percent weight loss) and increase the risk of adverse side effects, including nausea, vomiting, headache, constipation, confusion and dizziness, impaired alertness and motor coordination, worsening of depression or suicidal thinking, complex sleep behaviors, sleep paralysis, and compromised respiratory function.
  • a primary treatment for obesity includes dieting and physical exercise. The combination of dieting and exercising, however, rarely produces sustained weight loss, generally resulting from slow weight regain over time.
  • Altered eating has been recommended as a behavioral change to increase weight loss.
  • CDC recommends a variety of lifestyle interventions for weight loss, including: calorie restriction; time restricted feeding, where meals are consumed within a limited time window (ie., 6-8 hours) during the day; altemate-day fasting, where food consumption is unrestricted every other day and minimal or no calories are consumed on the other days; “5:2” eating patterns, where meals are unrestricted for 5 days each week, followed by 2 days of restricted calorie intake; and periodic fasting, where calorie intake is restricted for several days in a row (ie., 5 days) once per month, and food consumption is unrestricted on the other days.
  • Increased exercise is recommended as a behavioral change to increase and sustain weight loss.
  • the American College of Sports Medicine (ACSM) and CDC Guidelines propose increasing exercise to augment weight loss. For example, the Guidelines indicate that healthy adults aged 18-65 years should participate in moderate intensity aerobic physical activity a minimum of 30 min on five days per week, or vigorous intensity aerobic activity for a minimum of 20 minutes on three days per week.
  • weight loss is accompanied by persistent endocrine adaptations that cause an increase in appetite and decrease satiety, and there can be a physiological downregulation of pathways associated with weight loss and metabolism, thereby resisting continued weight loss and conspiring against long-term weight maintenance. This can lead to more robust weight regains as the weight lost progresses further from the “weight thermostat” set by years of overeating and all the offsetting mechanisms being activated.
  • Obesity is a chronic degenerative disease that can stem from the rewiring of biological mechanisms that hinder weight loss and promote weight gain.
  • lifestyle and behavioral interventions provide moderate efficacy, these strategies are limited by complications in adherence. They can be augmented by surgical intervention and/or pharmacological approaches (Muller TD. et al. Nat Rev Drug Discov 21(3):201-223 (2021)). Bariatric surgery generally is considered the most effective approach to weight loss, but it is expensive and limited to single patients, lacking the reach necessary to treat obesity at a global scale. Pharmacological agents can offer such treatment.
  • FDA-approved monotherapy options include phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phentermine-topiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave®) and semaglutide (sold under the trademark Wegovy®) medications.
  • These medications, and others can target components of the central nervous system or peripherally, such as in the gastrointestinal system, including, but not limited to the stomach, small intestine and colon, and pancreas (see e.g., FIG. 2).
  • monotherapies have limited efficacy, in part due to the metabolic redundancies and recruitment of alternate and counter- regulatory pathways, and desensitization.
  • Obesity is a disease with multiple etiologies; as described herein can be treated with a multi -targeted approach.
  • a multi-target approach provides greater benefit than any single medication alone.
  • a multi-targeted approach includes a combination therapy or a rotational combinatorial therapy.
  • a number of medications have been administered for long-term use for weight loss; these medications, include, but are not limited to, phentermine-topiramate, orlistat, lorcaserin, naltrexone-bupropion, and liraglutide. Treatment with these medications as monotherapies results in weight loss after one year that ranges from 3.0 to 6.7 kg (6.6-14.8 lbs) compared to placebo (Heymsfield et al., (2017) The New England Journal of Medicine 376 (3):254- 266). Information on how these drugs affect longer-term complications of obesity, such as cardiovascular disease or death is sparse. Obesity drugs do not target weight loss per se, but on appetite suppression and/or are label expansions of other marketed drugs.
  • semaglutide sold under the trademark Wegovy®
  • Thegovy® is a therapy designed for treatment of type-2 diabetes.
  • An effect of the drug was weight loss and it has been rebranded for weight loss treatment.
  • the combinatorial therapy can include a combination of therapeutics known to effect weight loss and/or pharmaceuticals previously used as weight loss monotherapies and dual therapies. Medications that previously were used for non-weight loss indications that exhibit weight loss as a secondary effect also can be included in the combination therapies herein.
  • the medications listed herein, such as, for example, below can be included in the combinations herein for treating overweight, obesity and/or for weight loss.
  • Pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and/or therapeutics for the combination therapy, such as therapeutics listed below.
  • Combinatorial regimens employing combinations, such as three or more of the medications below can be included in the combinations for weight loss, and/or treatment of overweight or obesity.
  • These drugs can be part of a rotational combinatorial regimen as well, in which two or more of the drugs are combined and administered for a predetermined time, and then are rotated with another drug or combination of drug for a predetermined time.
  • Amphetamines e.g., phentermine-topiramate
  • Amphetamines stimulate norepinephrine release that can result in increased blood pressure, heart rate, and cardiac excitability via binding to vasculature and heart adrenergic receptors. These drugs belong to the class of drugs known as sympathomimetic amines.
  • Amphetamines used as anti-obesity medication include, but are not limited to Methamphetamine (Desoxy ephedrine), Phenmetrazine (sold under the trademark Preludin®), Phendimetrazine, Phentermine, benzphetamine and Diethylpropion.
  • Phentermine sold under the trademark Adipex-P®
  • Diethylpropion sold under the trademark Tenuate®
  • Phentermine sold under the trademark Adipex-P®
  • Diethylpropion sold under the trademark Tenuate®
  • Several clinical studies report the absence of major adverse effects of phentermine or diethylpropion on blood pressure and heart rate; nonetheless, their use is contraindicated in patients with hypertension or elevated risk for cardiovascular disease (Muller etal., Nat Rev DrugDiscov 21 : 201-223 (2022)). Patients also quickly develop a tolerance to these medications.
  • Phentermine is a weight loss medication approved for short term use, such as less than a month (Hendricks et al., Obesity 17: 1730-35 (2009)). Patients can rapidly develop a tolerance to the medication. Amphetamines can be used in combination with other medications for treatment of overweight and obesity. For example, phentermine is used in combination with topiramate (combination sold under the brand name Qsymia®), which can be used for a longer period of time than phentermine alone. Previous studies show that the combination therapy results in an average of 5-10% weight loss, where weight loss corresponds to dosage. These can be administered in combination with liposomes provided herein, or incorporated into the liposomes. b. Lipase inhibitors (e.g., Orlistat)
  • Lipases are a class of digestive enzymes produced in the pancreas, mouth, and stomach to metabolize fat and to facilitate nutrient uptake.
  • Orlistat (sold under the trade name Xenical®) is a lipase inhibitor that reduces the uptake of dietary fat in the gastrointestinal tract. The resulting fat malabsorption after treatment with lipases facilitates a negative energy state leading to a placebo-normalized weight loss in the range of 2.5%.
  • Orlistat has shown beneficial effects on blood glucose, non-alcoholic fatty liver disease, and blood pressure (Muller et al. (2016) Pharmacol. Rev. 70:712- 746; Khera et al. (2016) Gastroenterology 154'.1309-1319. e7). The most common adverse events after orlistat treatment are of a gastrointestinal or digestive nature.
  • Serotonergic agonists - Neuromodulators e.g., lorcaserin
  • Serotonergic agonists act as anti-obesity medication by suppressing appetite through the activation of serotonin receptors.
  • Clinical serotonergic agonists include Fenfluramine, Dexfenfluramine, Sibutramine, Lorcaserin (sold under the trademark Belviq®), and Tesofensine. Cardiovascular safety concerns accompany Fenfluramine, Dexfenfluramine, and Sibutramine (Muller et al. (2016) Pharmacol. Rev.70 712- 746). The FDA requested withdrawal of Lorcaserin due to clinical trials showing an increased occurrence of cancer (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)).
  • Tesofensine an inhibitor of norepinephrine, serotonin, and dopamine reuptake that was initially advanced for treatment of Alzheimer disease.
  • a phase II study it was reported to dose-dependently decrease body weight by 4.4-10.4% (Wharton et al. (2015) J. Curr. Cardiol. Rep. 17:35,' Astrup A. et al. (2008) Lancet 372:1906-1913).
  • Bupropion/Naltrexone such as the product sold under the trademark Contrave® Naltrexone, an opioid antagonist, blocks the inhibitory effects of opioid receptors activated by P-endorphin released in the hypothalamus, which stimulates feeding.
  • Naltrexone reduces food intake in combination with bupropion, reuptake inhibitor of dopamine and norepinephrine. Naltrexone does not cause weight loss in monotherapy; patients treated with Naltrexone 32 mg plus Bupropion showed a body weight reduction of 6.1% after 56 weeks of treatment (Greenway et al. Lancet 376, 595-605 (2010)). No increased adverse cardiovascular events were noted in an analysis of a cardiovascular outcome trial after bupropion/naltrexone treatment (Nissen et al., JAMA 315:990-1004 (2016)). Another study showed that addition of a modified diet and increased exercise to the bupropion/naltrexone combination lost an average of 9.3% of their baseline body weight. e. Glucagon-like peptide-1 receptor (GLP1R) agonists
  • Glucagon-like peptide-1 acts at the pancreas to enhance the expression and secretion of insulin and to inhibit the release or glucagon.
  • GLP1 and glucose-dependent insulinotropic polypeptide are primary incretin hormones secreted from the intestine on ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic P cells.
  • GIP glucose-dependent insulinotropic polypeptide
  • Liraglutide (sold under the trademark Saxenda®), is a once-daily injectable GLPR1 agonist, and was the first GLP1R agonist approved for treatment of obesity. Liraglutide is short-acting, so it is administered daily. After one year of treatment, there was a reported mean decrease of 8% body weight in subjects treated with liraglutide compared to 2.6% in subjects treated with vehicle controls (Pi-Sunyer et al. (2015) N. Engl. J. Med. 373: 11-22). In 2020, treatment with 3 mg liraglutide was approved for weight management in adolescents with obesity.
  • GLP-1 increases insulin metabolism and plays a role in appetite and digestion. It is among the incretins, which are hormones released by the small intestine into the bloodstream following a meal to help lower blood sugar by triggering insulin and blocking other sugar sources. Medications that are GLP-1 agonists are referred to as incretin mimetics since they “mimic” the incretin hormone effects.
  • Semaglutide which is similar to and acts like a GLP-1 hormone, is used as an anti-diabetic medication. Semaglutide also slows down the rate at which food leaves the stomach (called gastric emptying). These actions cause a feeling of fullness, lowering appetite and resulting in weight loss. Semaglutide (sold under the brand name Wegovy®) is an injectable GLPR1 agonist FDA-approved in 2021 for chronic weight management in adults with obesity (URL: fda.gov/news-events/press- announcements/fda-approves-new-drug-treatment-chronic-weight-management-first- 2014).
  • Wegovy® weight loss medication is approved for treatment of adults with a body mass index (BMI) greater than or equal to 30mg/kg 2 alone or 27 mg/kg 2 with at least one weight-related comorbidity (e.g., high blood pressure, high cholesterol).
  • BMI body mass index
  • Semaglutide administered at a dose of 2.4 mg lowers mean body weight to -12-15% after 68 weeks of treatment (relative to -2.4% in placebo controls) with once-weekly treatment (Wilding et al., N. Engl. J. Med. 384(11): 989-1002 (2021)).
  • a longer mechanism of action compared to other weight loss medications allows semaglutide to be administered weekly; semaglutide is the first once-weekly GLP-1 agonist approved for weight loss.
  • GLP1R agonists are in clinical development, including formulations designed for oral administration (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)).
  • GLP1R agonists in clinical development include Efpeglenatide (s a long-acting GLP-1RA that is a single amino acid-modified exendin conjugated to a fragment crystallizable region of human immunoglobulin 4 via a 3.4-kDa mini-poly ethylene glycol linker), semaglutide sold under the trademark Rybelsus®, Danuglipron (formerly PF-06882961), GLPR-NPA, and lotiglipron (formerly PF-07081532).
  • Efpeglenatide s a long-acting GLP-1RA that is a single amino acid-modified exendin conjugated to a fragment crystallizable region of human immunoglobulin 4 via a 3.4-kDa mini-poly ethylene glycol linker
  • semaglutide sold under the
  • GLP-1 receptor agonists approved to improve glycemic control and lower blood sugar in adults with type 2 diabetes exhibit a secondary effect of increased weight loss.
  • Trulicity® Triggery®
  • UCP1 Uncoupling Protein 1
  • Small molecule uncouplers for antiobesity application include 2,4-dinitrophenol (DNP) and BAM15 ((2- fhiorophenyl) ⁇ 6-[(2-fluorophenyl)amino]( 1 ,2, 5 -oxadiazolo [3 ,4e] pyrazin-5-yl) ⁇ amine).
  • DNP is the first small molecule uncoupler tested clinically, but has limited utility owing to toxicity. Controlled-release oral formulation of DNP are under investigation to achieve an enhanced therapeutic index (Muller el al.. Nat Rev Drug Discov 21 : 201-223 (2022)).
  • BAM15 is an orally administered therapeutic that reverses diet-induced obesity and insulin resistance in mice (Alexopoulos et al., Nat. Commun. 11(1): 2397 (2020)).
  • Thyroid hormone can decrease body weight and body fat by stimulating energy expenditure (Muller et al., Pharmacol. Rev. 70:712-746 (2016)). Thyroid hormone also can improve hepatic lipid metabolism and decrease low-density lipoprotein (LDL) cholesterol via enhanced reverse cholesterol transport and clearance of LDL via the liver (Baxter et al., Nat Rev Drug Discov 8:308-320 (2009)).
  • LDL low-density lipoprotein
  • the biologically active form of thyroid hormone is tri-iodothyronine (T3), which promotes its pharmacology through two specific nuclear thyroid receptor (TR) isoforms, TRa and TRp.
  • T3 increases metabolic rate in a variety of species, including mice, rats, and humans (Muller et al., Pharmacol. Rev.
  • T3 modulation of metabolic rate includes uncoupling of oxidative phosphorylation from mitochondrial ATP synthesis in skeletal muscle and other peripheral tissues, regulation of lipogenesis, activation of Na+/K+ ATPase, enhanced mitochondrial biogenesis, and stimulation of futile cycling (Muller el al.. Pharmacol. Rev. 70:712-746 (2018)).
  • Excess thyroid hormone also leads to muscle and bone catabolism, as well as several cardiovascular adverse effects including cardiac arrhythmia, tachycardia, and heart failure, severely limiting its use as a weight loss therapeutic. h. Drug cocktails
  • Clarkotabs developed in 1941, were among the first commercially distributed combination diet pills; the combination sought to harness the anorectic effect of amphetamines with the thermogenic effect of thyroid hormone, with Aloin and Atropine sulfate to counteract potential adverse cardiovascular effects (Muller et aL, Pharmacol. Rev. 70:712-746 (2016)).
  • d- amphetamine or related analogs e.g., Diethylpropion, Fenfluramine, Sibutramin, or Fenproporex
  • thyroid hormones diuretics
  • laxatives Chlorthalidon, Ephedrine, and/or Phenolphthalein.
  • Substances such as Digitalis, Belladonna, benzodiazepines, barbiturates, corticosteroids, cardiac glycosides, beta-blocker, and potassium were common additives used to counteract or mask adverse cardiovascular effects of the drug cocktail (Cohen PA. et al. , Am J Public Health 102: 1676-1686 (2012)).
  • Topiramate is a sulfamate-substituted monosaccharide derived from D-fructose, commonly used to treat epilepsy and migraine. Although the mechanism of how the combination improves systemic metabolism is unknown, placebo-normalized weight loss ranges from 5.9%-9.6% (Muller et al., Pharmacol. Rev. 70:712-746 (2016)).
  • the weight loss combination of the medications naltrexone and bupropion combines two medications approved for indications other than weight loss.
  • Bupropion a dopamine-norepinephrine reuptake inhibitor is approved to treat depression and aid in smoking cessation
  • naltrexone an opioid receptor antagonist is approved to treat alcohol and opioid dependence.
  • the naltrexonebupropion combination is marketed under the trade name Contrave® and is FDA approved for chronic weight management. i. Cannabinoid receptor antagonists
  • Rimonabant an endocannabinoid 1 receptor (CB1) antagonist, acts by modulating neurons in both homeostatic and hedonic feeding circuits, leading to placebo- subtracted weight loss of ⁇ 2.6-6.3 kg (Muller etal., Pharmacol. Rev. 70: 712-746 (2016); Pi-Sunyer et al. JAMA 295:761-775 (2006)). Rimonabant was discontinued in 2009 due to serious adverse psychiatric effects (Sam et al., J Obes 2011 :432607 (2011)). j. GIPR agonists and GIPR/GLP1R combination agonists
  • Glucose-dependent insulinotropic polypeptide is a hormone involved in blood sugar control.
  • GIP receptor (GIPR) agonists have been shown to decrease food intake, increase energy expenditure, decrease body weight and to improve glucose handling in preclinical studies (Mroz et al., Mol. Metab. 20:51-62 (2019); Zhang et al., Cell Metab. 33:833-844.e5 (2021)).
  • GIP is known to be upregulated following Rou-en-Y gastric bypass.
  • Body weight loss associated with GLP-1 agonist treatment is enhanced when GLP-1 and GIP are co-administered (Matthias Tschbp oral presentation at ADA (American Diabetes Association), 2011; Tschbp et al., Diabetes 66: 1766-1769 (2017)).
  • GIPR/GLP1R dual agonists and co-admini strati on of GLP-1R agonists and GIPR agonists have demonstrated metabolic benefits and reduced body weight in mice when compared to GLP1R agonists (Coskun et al., Mol. Metab. 18: 3-14 (2016); Finan et al., Sci. TranslMed. 5, 209ral51 (2013)).
  • GIP can also enhance GLP-1 -mediated body weight loss.
  • GIP/GLP1 dual agonists include Tirzepatide, GIP/GLP peptide I, GIP/GLP peptide II, and NN9709.
  • Tirzepatide is a combination medication that activates the GLP-1 and GIP receptors; tirzepatide contains GLP-R and GIPR agonists).
  • Tirzepatide (available under the trademark Mounjaro®) is a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist. The drug is manufactured by Eli Lilly & Co. and was approved in May 2022. Tirzepatide works similarly to the other drugs in the GLP-1 receptor agonist family, but it has additional effects that appears to give it a slight edge.
  • GLP-1 Glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • Tirzepatide is a 39 amino acid peptide GLP-1 receptor agonist based on the GIP sequence. It contains 2 noncoded amino acids in positions 2 and 13, a C-terminal amide, and a lysine residue at position 20 that is attached to a 1, 20 eicosanoic acid via a linker.
  • Studies of tirzepatide were conducted through two placebo-controlled trials (SURP ASS-1 and -5), three trials in combination with metformin, sulfonylureas and/or SGLT2 inhibitors (SURPASS-2, -3, -4), and two additional trials conducted in Japan.
  • Tirzepatide (sold under the trademark Mounjaro®) was approved by the US Food and Drug Administration as a once-weekly subcutaneous injection to treat adults with type 2 diabetes, to improve blood sugar levels. As detailed above, and herein, Tirzepatide showed significantly better long-term blood sugar control (i.e., A1C) and weight loss compared to semaglutide.
  • A1C long-term blood sugar control
  • Glucagon and glucagon agonists have been shown to promote satiety and to increase energy expenditure when used in combination in both rodents and human (Kleinert et al., Int J Mol Set. 20(21): 5407 (2019)).
  • a triagonist peptide that act as an agonist of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors was previously described to effect weight loss in animal models (Finan et al., Nat Med 21(l):27-36 (2015)).
  • GLP-1 receptor (GLP1R) agonists in combination with glucagon (GcgR) agonists participate in several mechanisms of body weight reduction, including appetite suppression, thermogenesis and lipolysis, while minimizing the risk of hyperglycemia (Muller et al., Nat Rev Drug Discov 21 (3):201 -223 (2021)).
  • Combination strategies of these agonists have included Cotadutide (MEDI0382), BI 456906, Efinopegdutide ( LAPS GLP/GCG), and oxyntomodulin (OXM; SEQ ID NO:4).
  • Several GIP/GLP1 /glucagon tri-agonists also have been developed for weight loss, including HM15211 ( LAPS Triple Agonist), GGG tri-agonist, LY3298176 and NN9423.
  • the field of pharmaceutically assisted weight loss has focused on monotherapy and the most effective pharmaceuticals include versions of a GLP-1 agonist.
  • a GLP-1 agonist Although approved for use in human, several therapeutics that show efficacy are not widely used because of adverse side effects or toxicity associated with administration of the medications at the therapeutic or FDA approved dosages.
  • patients taking Rimonabant experience adverse psychiatric effects
  • patients taking thyroid hormones and amphetamines experience adverse cardiac effects
  • patients taking lorcaserin show increased occurrence of cancer
  • patients taking orlistat experience gastrointestinal issues.
  • a combination of therapeutics administered in accord with the rotational protocol/regimen described herein incorporates pharmacological principles known to occur after a gastric bypass, as well as overcoming tolerance and/or desensitization to medications due to receptor downregulation/desensitization to medications.
  • the combination therapy can be administered at a lower dosage than the monotherapeutic dosage to decrease side effects associated with administration of the monotherapy.
  • the combination therapy is rotated at intervals (ie., monthly, bi-monthly, or trimonthly) wherein the patient is not administered an individual therapeutic for an extended period; the finite time period for administration of the medication decreases the time for developing adverse side effects (z.e., severe adverse effects) or downregulation of the pathways activated by the medications.
  • the rotational combinatorial therapy described herein can include a combination of a plurality of different known therapeutics and/or treatments.
  • the combination for treatment of obesity or for weight loss can include more than one previously characterized weight loss treatment.
  • the rotational combinatorial therapy can include one or more FDA-approved monotherapy options, such as, for example, phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phenterminetopiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave ®), tirzepatide (sold under the trademark Mounjaro®), and semaglutide (sold under the trademark Wegovy®) medications.
  • FDA-approved monotherapy options such as, for example, phentermine (sold under the trademark Adipex-P®),
  • the rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, where a combination of therapeutics can be developed for treating the disease or disorder or condition.
  • a treatment of obesity or overweight for which a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways are known to be involved in disease progression;
  • Bariatric procedures include, for example, laparoscopic adjustable gastric banding, Roux-en-Y gastric bypass, vertical-sleeve gastrectomy, and biliopancreatic diversion.
  • Surgery for severe obesity is associated with long-term weight loss, improvement in obesity -related conditions, and decreased overall mortality.
  • a resolution of co-morbidities, such as diabetes, hypertension, fatty liver, urinary distress, and hyperthyroidism, also have been shown to occur following bariatric surgery (Susmallian et al., Medicine 98(3): el3824 (2019)).
  • Weight loss of between 12% and 35% (depending on the type of procedure performed) is expected at 1-2 years after surgery.
  • the average weight loss in clinical practice after 2 years for adjustable gastric banding is about 20%
  • weight loss after Roux-en-Y gastric bypass is about 30%
  • weight loss after biliopancreatic diversion or duodenal switch is about 35% (Cummings et al., (2004) J Clin Endocrinol Metab 59:2608-2615). Complications occur in about 17% of cases and reoperation is needed in 7% of cases (Chang etal., JAMA Surgery (Meta-analysis, Review) 149 (3): 275- 87 (2014); doi: 10.1001/jamasurg.2013.3654).
  • Weight loss after bariatric surgery results, not only from the bypass and other physical changes, but also from factors other than the physical changes to the gastrointestinal tract and calorie reduction following surgery (Cummings et al. , J Clin Endocrinol Metab 89:2608-2615 (2004)).
  • Post-surgical hormonal alterations occur that can change activation of brain pathways that regulate appetite cues (Batterham et al., Nature 450: 106-109 (2007)).
  • Vagal nerve stimulation delivers short bursts of energy into the vagus nerve in the brain, is used for treatment of drug-resistant epilepsy, and has been associated with weight loss.
  • VNS has been associated with weight loss (> 5% of body weight) within 6-12 months of stimulation.
  • the weight loss is due to the action of afferent fibers of the nerve which participate in the brain-gut axis, which take part in a feedback loop induced by the presence of food in the gastrointestinal tract inducing hypometabolism of the hypothalamus and subsequent involvement of the satiety centers, thereby controlling food intake.
  • the benefits of surgical intervention can be achieved by applying the combinatorial, and rotational combinatorial methods provided and described herein for weight loss by selecting and administering combinations of drugs/treatments to mimic the hormonal and physiological effects of surgery.
  • the combinations of therapeutics also can be rotated.
  • Small molecules drugs for use for weight loss and other indications are set forth in the following table: These small molecule drugs can be formulated as part of the liposomes (inside and outside) or administered as a separate dosage from the liposomes provided herein, where the liposomes display and/or contain one or more peptide drugs.
  • the combinatorial therapy and rotational combinatorial therapies provided herein correct excess weight while reducing risk for adverse effects (z.e., cardiovascular and psychological adverse effects) and without increasing muscle loss by taking into account the multifactorial processes associated with metabolism and weight gain.
  • Combinations for use in the combinatorial and rotational combinatorial therapies provided herein include a plurality of medications and/or treatments.
  • the combinations can be rotated to overcome downregulation that occurs after prolonged monotherapy.
  • the combinations target the multifactorial processes involved in weight gain or failure to lose weight.
  • the rotation prevents or mitigates the desensitization/downregulation that occur upon long-term exposure to some of the weight loss therapeutics.
  • the rotational combinatorial therapy herein includes combinations of medications that: act peripherally to impaired dietary fat absorption; act centrally to decrease food intake; facilitate energy expenditure; and increase muscle development.
  • the combinatorial, and the rotational combinatorial therapies described herein for treating overweight or obesity also can decrease risk for obesity-related comorbidities including metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), cancer, elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
  • diabetes e.g., diabetes mellitus type 2
  • cardiovascular disease e.g., heart attack, stroke
  • cancer elevated blood pressure
  • elevated blood cholesterol elevated blood cholesterol
  • triglyceride levels and others.
  • the combination and rotational combinatorial therapy described herein is not associated with the harmful side effects associated with surgery, such as high cost, and the risk of developing intra-abdominal abscess, thrombosis, dehydration, and type 1 diabetic ketoacidosis.
  • Developing a combinatorial therapy and rotational combinatorial therapy for weight loss includes assessing a variety of factors, selecting patients for treatment, selecting molecular and/or cellular pathways for treatment, selecting therapeutics for treatment, and developing the rotational regimen, including timing for administration and timing for rotating the combinations, and dosages.
  • molecular and/or cellular pathways known to contribute to weight loss or appetite regulation or metabolism, or molecular and/or cellular pathways that are associated with weight loss or appetite regulation or metabolism can be targeted for therapy.
  • peptides or proteins that are associated with molecular and/or cellular pathways that contribute to or impact weight loss, appetite regulation, and/or metabolism can be selected for use for treatment of overweight or obesity, or to effect weight loss.
  • the combinations of treatments are identified based on these criteria and those discussed below to combine treatments that, for example, mimic the effects of bariatric surgery.
  • the combinations (or clusters) can be rotated to produce rotational combinatorial protocols to avoid desensitization and/or receptor downregulation.
  • peptides known to contribute to weight loss and/or appetite regulation and/or metabolism can be assessed and included in the combinations.
  • pathways to target for weight loss and peptides to include in the combinations the following can be assessed: 1) the pharmacodynamic and pharmacokinetic properties of each peptide individually and in combinations with other peptides and/or other therapeutics; 2) stability in serum, to ensure adequate stability for the intended use; 4) solubility of the peptides and/or other therapeutics; the delivery profile to ensure activity, stability, solubility and other characteristics to ensure therapeutic activity and efficacy.
  • Homeostatic mechanisms involve multiple components including neuronal circuits, some originating in hypothalamus and brain stem, as well as peripherally derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components can result in or contribute to obesity.
  • Type 2 diabetes symptoms resolve quickly after surgery and before weight loss, and patients exhibit improved glucose homeostasis after RYGB compared to similar weight loss achieved via other means (z.e., diet and/or exercise). These rapid changes indicate that the resultant weight loss and/or reduced caloric intake, does not explain these weight-independent antidiabetic actions of RYGB.
  • Hormone profiling of subjects after bariatric surgery provides the following insights: 1) weight loss is an integrated hormonal process, 2) multiple peptides are involved (Combination) and 3) peptides have fluctuations and in timing and release (Rotation).
  • peptides and/or other therapeutics including peptide/receptor agonist or antagonists, depending on the target, in combinations that mimic the fluctuations of peptides whose expression or activity is altered after gastric bypass or other weight loss surgery.
  • the combination therapy herein takes into account the integrated processes that occur during weight loss, and seek to mimic the multiple pathways using combinations of therapeutics administered to mimic the fluctuations in timing and release that occur following gastric bypass.
  • a combinatorial therapeutic regimen is described because multiple pathways are involved and because monotherapies are generally ineffective or less effective than gastric bypass.
  • the combination therapies described herein administered with a rotational approach overcome desensitization to the peptides and/or therapeutics that can occur due to receptor downregulation, which occurs over time after monotherapy or long-term combination therapy.
  • Weight loss surgery patients also experience an increase in muscle mass following surgery; therapeutic agents that alter pathways that affect (z.e., increase) muscle generation to increase muscle mass during the weight (fat) loss protocol. Weight loss surgery patients also experience a decrease in muscle mass following surgery, and it is provided herein to provide such patients with agents that alter pathways that affect (z.e., increase) muscle generation to increase muscle mass during the weight (fat) loss protocol.
  • Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes.
  • the therapy described herein can include peptides to decrease insulin resistance that can be a comorbidity with overweight or obesity.
  • the combinations can include peptides that are associated with metabolism, satiety, physiological response to hunger or food ingestion, and/or digestion. These combinations include peptides or therapeutics that target pathways altered following bariatric surgery.
  • the combinations can include therapeutics that impact homeostatic energy balance, including neuronal circuits, some originating in the hypothalamus and brain stem, as well as peripherally derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components results in obesity.
  • the hormonal signaling network that provides the brain input related to metabolic status and energy stores includes leptin, insulin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), peptide YY3-36 (PYY3-36), and ghrelin. Any of the peptides, proteins, and pathways detailed below can be targeted for the combination and/or combination rotational therapy described herein.
  • the combinatorial therapies and rotational combinatorial therapies provided herein target a plurality thereof.
  • Components of these pathways can be included in the combination and/or rotational combinatorial therapy to effect weight loss, or to treat or ameliorate comorbidities of overweight or obesity.
  • GLP-1 The sequence of GLP-1 is set forth in SEQ ID NO: 1, and the chemical formula is C186H275N51O59.
  • the glucagon-like peptide-1 (GLP-1) is released by L- cells in response to food ingestion. Simulation of L-cells increases both GLP-1 and GLP-1 related peptides, such as glicentin, oxyntomodulin intervening peptide-2 and GLP-2.
  • Agents described for use in the rotational combinatorial treatment herein can work by activating the Glucagon-like peptide- 1 receptor (GLP-1R) or by inhibiting the breakdown of GLP-1 like dipeptidyl peptidase- 4 (DPP-4) inhibitors (see Bruton, ., Int. J of Clin Pract.
  • DPP-1 inhibitors and their uses are previously described (see e.g., WO 2002/068420, WO 2004/018467, WO 2004/018468, WO 2004/018469, WO 2004/041820, WO 2004/046148, WO 2005/051950, WO 2005/082906, WO 2005/063750, WO 2005/085246, WO 2006/027204, WO 2006/029769, W02007/014886; WO 2004/050658, WO 2004/111051, WO 2005/058901, WO 2005/097798; WO 2006/068163, WO 2007/071738, WO 2008/017670; WO 2007/128721, WO 2007/128724, WO 2007/128761, or WO 2009/121945).
  • Glucagon-like peptide- 1 receptor agonists can be formulated as therapeutics in the combinational therapy provided herein.
  • a GLP-1R agonist can be formulated in combination with additional receptor agonists (i.e., GIP and/or glucagon receptor agonist(s)).
  • additional receptor agonists i.e., GIP and/or glucagon receptor agonist(s)
  • a single biagonist or triagonist peptide in the combinatorial rotational therapy herein can act as an agonist for multiple receptors of the peptides, proteins and/or pathways detailed above, or pathways involving hunger, digestion and/or metabolism.
  • a biagonist or triagonist peptide that acts as an agonist of one or more glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors, such as the previously described triagonist peptide can be included in the combination therapy to effect weight loss (Finan et al., Nat Med 21(l):27-36 (2015)).
  • GLP-1 glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • glucagon receptors such as the previously described triagonist peptide
  • Adiponectin is a 244-amino-acid-long peptide (SEQ ID NO:2), protein hormone and adipokine, involved in regulating glucose levels as well as fatty acid breakdown (Iglesias, J. J., European Journal of Endocrinology 148 (3): 293- 300). Adiponectin is secreted from adipose tissue into the bloodstream and is abundant in plasma relative to several other hormones. High adiponectin levels correlate with a lower risk of diabetes mellitus type 2 (Li et al., JAMA 302(2): 179- 188 (2009)).
  • Adiponectin plays a role in suppressing metabolic derangements that can result in type 2 diabetes, obesity, atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and an independent risk factor for metabolic syndrome (Ukkola et al., J Mol Med 80 (11): 696-702 (2002); Iglesias, J. J., European Journal of Endocrinology 148 (3): 293-300).
  • Adiponectin in combination with leptin has been shown to completely reverse insulin resistance in mice (Chen et al., Diabetologia 49 (6): 1292-302 (2006)).
  • Adiponectin enhances insulin sensitivity primarily though regulation of fatty acid oxidation and suppression of hepatic glucose production (Li et al., JAMA 302 (2): 179 (2009)). Adiponectin exerts weight reduction effects via the brain, similar to the action of leptin; and adiponectin and leptin can act synergistically (Coppola et al., International Journal of Cardiology. 134 (3): 414-6 (2009)).
  • Adiponectin self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer, and the trimers continue to selfassociate and form hexamers or dodecamers. Studies showed that the high-molecular weight form of adiponectin is the most biologically active form regarding glucose homeostasis (Kuo et al., IntJ of Obesity 35 (12): 1487-94 (2011)).
  • Plasma levels of adiponectin are lower in obese subjects than in lean subjects (Ukkola et al., J Mol Med 80 (11): 696-702 (2002)).
  • Adiponectin is secreted into the bloodstream where it accounts for approximately 0.01% of all plasma protein at around 5-10 pg/mL (mg/L).
  • Leptin is a 167 amino acid hormone (SEQ ID NO:3) predominantly made by adipose cells and enterocytes in the small intestine that helps to regulate energy balance by inhibiting hunger, which in turn diminishes fat storage in adipocytes. Leptin acts on cell receptors in the arcuate and ventromedial nuclei, as well as other parts of the hypothalamus and dopaminergic neurons of the ventral tegmental area, consequently mediating feeding (Brennan et al. , Nature Clinical Practice. Endocrinology & Metabolism (6): 318-27 (2006); Bouret el al..
  • POMC arcuate proopiomelanocortin
  • leptin inhibits hunger by 1) counteracting the effects of neuropeptide Y, a potent hunger promoter secreted by cells in the gut and in the hypothalamus, and 2) counteracting the effects of anandamide, another potent hunger promoter that binds to the same receptors as THC (Elias et al., Neuron. 23 (4): 775-86 (1999)).
  • leptin stimulates satiety by promoting the synthesis of a-MSH, a hunger suppressant (Fekete et al., JNeurosci 20 (4): 1550— 8 (2000)).
  • leptin signals to the brain in proportion to the amount of fat that is stored in the body.
  • leptin reduces appetite as a circulating signal
  • obese individuals generally exhibit a higher circulating concentration of leptin than normal weight individuals due to their higher percentage body fat (Considine et al., The New England Journal of Medicine 334 (5): 292-295 (1996)).
  • Leptin also reduces appetite in response to feeding, but obese people develop a resistance to leptin, similar to resistance of insulin in type 2 diabetes patients, where elevated levels fail to control hunger and modulate weight in obese people.
  • leptin resistance is changes to leptin receptor signaling, particularly in the arcuate nucleus, however, deficiency of, or major changes to, the leptin receptor itself are not thought to be a major cause.
  • Triglycerides crossing the blood brain barrier (BBB) can induce leptin and insulin resistance in the hypothalamus (Fomy-Germano et al., Front in Neurosci 12: 1027 (2019)). Triglycerides can also impair leptin transport across the BBB. id. Leptin interacts with amylin, a hormone involved in gastric emptying and creating a feeling of fullness.
  • Leptin can be a target for developing a therapeutic, in combination with any of the peptides or therapeutics described herein, or known to be involved with metabolic pathways, in accord with a rotational regimen, such as, for example, to mimic satiety.
  • Oxyntomodulin is a 37-amino acid peptide (SEQ ID NO:4) hormone produced by the oxyntic (fundic) cells of the oxyntic (fundic) mucosa in the colon.
  • Oxyntomodulin suppresses appetite; the mechanism by which it does so is not understood. It binds to the GLP-1 receptor and to the glucagon receptor, but it is not known whether the effects of the hormone are mediated through these receptors or through an unidentified receptor.
  • Oxyntomodulin has been investigated as a blood-glucose regulating agent in connection with diabetes. Oxyntomodulin is a candidate for treating obesity because of its ability to suppress appetite (Shankar et al., Diabetes. 67 (6): 1105-1112 (2018)). In a 4-week study, healthy overweight and obese subjects treated with oxyntomodulin injections had an average weight loss of 2.3 ⁇ 0.4kg compared to those treated with saline who had an average of 0.5 ⁇ 0.5kg (Wynne et aL, Diabetes. 54 (8): 2390-2395 (2005)).
  • Sermorelin acetate is peptide analog of growth hormone- releasing hormone (GHRH; see SEQ ID NO:29) which is used as a diagnostic agent to assess growth hormone (GH) secretion to diagnose growth hormone deficiency.
  • Sermorelin acetate is a 29-amino acid peptide representing the 1-29 fragment from endogenous human GHRH, thought to be the shortest fully functional fragment of GHRH (Prakash et al., BioDrugs 12(2): 139-57 (1999); Rang, Dale, Ritter & Moore. Pharmacology. Edinburgh, Churchill Livingstone (2005)).
  • Peptide YY is a short (36-amino acid) peptide released from cells in the ileum and colon in response to feeding. In the blood, gut, and other elements of periphery, PYY acts to reduce appetite; similarly, when injected directly into the central nervous system, PYY is also anorexigenic, i.e., it reduces appetite (Woods et al., J Clin Endocrinol Metab. 93 (11 Suppl 1): S37-50 (2008)).
  • PYY The two major forms of peptide YY (PYY) are PYYI-36 and PYY3-36 (SEQ ID NO:37), which have PP fold structural motifs.
  • PYYi 36 is rapidly cleaved by DPP -IV to its major active form, Peptide YY3-36.
  • PYY3-36 is a linear peptide that contains 34 amino acids with structural homology to NPY and pancreatic peptide (Murphy et al., Nature 444 (7121): 854-59 (2006)).
  • PYY3-36 is the most common form of circulating PYY, which binds to the NPY receptor type 2 (Y2R).
  • This receptor is highly expressed in parasympathetic and sympathetic neurons of the periphery as well as in several regions of the CNS, including the limbic and cortical areas and the brainstem (Stadlbauer et al. , Neuroendocrinol. 38: 1-11 (2015)).
  • Oxyntomodulin and peptide tyrosine-tyrosine (PYY) are released from intestinal enteroendocrine cells following a meal. These circulating hormones are considered to be satiety signals, as they decrease food intake, body weight, and adiposity in rodents (Wynne et al.. Nat Clin Pr act Endocrinol Metab. 2(11), 612-20 (2006)).
  • PYY The anorectic effect of PYY is a candidate for a future obesity drug (Murphy et al., Nature (2006) 444 (7121): 854-59). Attempts to use PYY directly as a weightloss drug have met with some success (Bartolome et al., Obes Surg. (2002) 12(3):324- 7). PYY decreases food intake and body weight in rodents and humans (Batterham et al., Nature (2002) 418: 650-654; Batterham et al., N. Engl. J. Med. (2003) 349:941- 948).
  • Amylin The Amylin peptide hormone is co-secreted with insulin from the pancreatic P-cells at a ratio of approximately 100: 1 (insulimamylin) into the blood circulation and is cleared by peptidases in the kidney, so it does not occur in the urine (Higham et al., Eur. J. Biochem. 267 (16): 4998-5004 (2000)).
  • Amylin functions as part of the endocrine pancreas and plays a role in glycemic regulation by slowing gastric emptying and promoting satiety, thereby preventing post-prandial spikes in blood glucose levels.
  • Amylin's metabolic function is well-characterized as an inhibitor of the appearance of nutrients [especially glucose] in the plasma (Pittner et al., J. Cell. Biochem 55 Suppl: 19-28 (1994)).
  • the overall effect is to slow the rate of appearance (Ra) of glucose in the blood after eating; this is accomplished via coordinate slowing down gastric emptying, inhibition of digestive secretion [gastric acid, pancreatic enzymes, and bile ejection], and a resulting reduction in food intake. Appearance of new glucose in the blood is reduced by inhibiting secretion of the gluconeogenic hormone glucagon.
  • Amylin peptide analogs have been developed and include Pramlintide (see SEQ ID NO:38), Cagrilintide (SEQ ID NO:43), and ZP 8396.
  • a 2008 study reported a synergistic effect of a human leptin (metrelepin) and an amylin analog (pramlintide) for obesity treatment in diet-induced obese rats and human subjects by restoring hypothalamic sensitivity to leptin (Roth et al., PNAS 105(20):7257-62 (2008)).
  • Pramlintide is approved for patients with Type 1 and Type 2 diabetes in combination with insulin, metformin, or sulfonylurea.
  • Cagrilintide is a long-acting amylin analogue amenable for once-weekly dosing.
  • Dual-acting amylin and calcitonin receptor agonists can induce weight loss in pre-clinical models of obesity (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)).
  • Dual agonists include davalintide (AC2307; SEQ ID NO:27), KBP-088 (see SEQ ID NO:31), KBP- 089 (see SEQ ID NO:32), and KBP-042 (SEQ ID NO:42).
  • Tesamorelin is a 44 amino acid synthetic form of growth-hormone-releasing hormone (GHRH) which is used in the treatment of HIV-associated lipodystrophy, approved initially in 2010. It is produced and developed by Theratechnologies, Inc. of Canada. Tesamorelin is sold under the trademark Egrifta® medication.
  • GH binds with receptors present on various body organs and regulates body composition; the regulation is primarily due to the combination of anabolic and lipolytic mechanisms.
  • the main mechanisms by which tesamorelin reduces body fat mass are lipolysis followed by reduction in triglyceride levels (Benedini et al., BioDrugs. 22 (2): 101-12 (2008)).
  • Pancreatic polypeptide is a 36 amino acids long peptide (human sequence of mature PP set forth in SEQ ID NO:40 (APLEPVYPGD NATPEQMAQY AADLRRYINM LTRPRY) that regulates pancreatic secretion activities by both endocrine and exocrine tissues.
  • PP is synthesized as a 95 aa polypeptide precursor in the pancreatic islets of Langerhans.
  • PP also affects hepatic glycogen levels and gastrointestinal secretions.
  • PP secretion in humans increases after a protein meal, fasting, exercise, and acute hypoglycemia, and decreases after increased food intake and in patients with anorexia nervosa.
  • pancreatic polypeptide concentration is 80 pg/mL; and after meal consumption the levels rise to 640 to 800 pg/mL.
  • Glucose and fats also induce PP and increase the PP level; however, upon parenteral introduction, the hormone levels do not change.
  • Peripheral administration of PP has been shown to decrease food intake in rodents (Batterham et al., The Journal of Clinical Endocrinology and Metabolism. 88 (8): 3989-92 (2003)).
  • PP inhibits pancreatic secretion of fluid, bicarbonate, and digestive enzymes; stimulates gastric acid secretion; is the antagonist of cholecystokinin; and opposes pancreatic secretion stimulated by cholecystokinin (Washabau, Canine and Feline Gastroenterology. Philadelphia, PA, Saunders (2013)).
  • GIP2 Gastric Inhibitory Polypeptide
  • K cells which are found in the mucosa of the duodenum and the jejunum of the gastrointestinal tract.
  • This endogenous hormone known to be upregulated post Rou-en-Y gastric bypass.
  • GIP is thought to induce insulin secretion, which is stimulated primarily by hyperosmolarity of glucose in the duodenum which has led to referencing GIP as glucose-dependent insulinotropic peptide, while retaining the acronym ”GIP” (Thorens, Diabete & Metabolisme. 21 (5): 311 8 (2014)).
  • GIP is known to inhibit apoptosis of the pancreatic beta cells and promote their proliferation.
  • Enterostatin is a peptide previously shown to selectively reduce fat intake, lower cholesterol, reduce angiogenesis, and regulate analgesia. Enterostatin is produced in the gastric mucosa and epithelium of the small intestine, is absorbed from the digestive tract, and functions as a strong anorectic peptide to selectively decrease fat consumption in rodents (D.L. Nelson, D.R. Gehlert, in Comprehensive Medicinal Chemistry II, 2007). Enterostatin interacts with a variety of hormone systems and receptors (Charlotte Erlanson-Albertsson, in Handbook of Biologically Active Peptides (Second Edition), 2013).
  • Enterostatin knock-out mice showed significantly increased serum cholesterol compared to wild-type controls and administration of enterostatin and its peptide fragment DPR reduce serum cholesterol after oral administration in mice (Miller et al., Am J Physiol Endocrinol Metab. 297(4):E856-65 (2009); Takenaka et al., Biosci Biotechol Biochem. 67(7): 1620-22 (2003)).
  • VPDPR enterostatin protein sequences
  • APGPR human; SEQ ID NO: 18
  • VPGPR rat; SEQ ID NO: 19
  • Ghrelin (SEQ ID NO:23) is a stomach-derived peptide hormone that signals to the hypothalamus to stimulate homeostatic food intake and hunger; ghrelin levels increase before a meal and when a person is hungry, and levels decrease after a meal. Daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. Strategies to target Ghrelin include suppressing levels of the hormone or antagonizing its receptor, growth hormone secretagogue receptor (GHSR) (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). GHSR antagonists include antimicrobial peptide 2 (LEAP2).
  • GHSR growth hormone secretagogue receptor
  • ghrelin levels decreased and remained low for at least 5 years following surgery (Bohdjalian et al., Obes Surg 20:535-540 (2010)).
  • Fasting ghrelin also decreased after laparoscopic sleeve gastrectomy and postsurgical circulating ghrelin decreased following Roux-en-Y gastric bypass, compared to both obese and normal weight control subjects.
  • ghrelin pathway can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
  • CCK mediates satiety by acting on CCK receptors distributed throughout the central nervous system. In response to meal initiation, plasma CCK levels have been reported to rise within 15 minutes (Liddle et al., J Clin Invest. 75(4): 1144-1152 (1985)). CCK also has stimulatory effects on the vagus nerve, which can be inhibited by capsaicin (Holzer et al. , American Journal of Physiology. Gastrointestinal and Liver Physiology 275 ( 1 ) : G8-G13 (1998)). The stimulatory effects of CCK oppose those of ghrelin, which has been shown to inhibit the vagus nerve (Kobelt et al., American Journal of Physiology 288 (3): R751-8 (2005)).
  • CCK was the first gut hormone reported to affect appetite and has been shown to dose-dependently reduce food intake in both rats and humans (Gibbs et al., Nature. 245: 323-325 (1973); Lieverse et al., Gut. 36(2): 176-179 (1995)).
  • the effects of CCK vary between individuals, and are demonstrated in animal studies. For example, in rats, CCK administration significantly reduces hunger in adult males, but is less effective in younger subjects, and less effective in females. The hunger-suppressive effects of CCK also are reduced in obese rats (Fink et al.. Experimental Brain Research 123 (1-2): 77-83 (1998)).
  • CCK is predominantly synthesized and released from the duodenum and jejunum, where its local regulatory effects include stimulation of gallbladder contraction and inhibition of gastric emptying (Buffa et al.. Gastroenterology. 70(4): 528-532 (1967; Dufresne et al., Physiol Rev. 86(3): 805- 847 (2006)).
  • the mechanism for hunger suppression by CCK is thought to be related to a decrease in the rate of gastric emptying (Shillabeer et al., he American Journal of Physiology 252(2): R353-R360 (1987)).
  • CCK can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
  • Vasoactive intestinal peptide is an endogenous peptide hormone that is 28 amino acid residues.
  • VIP was shown to induce smooth muscle relaxation (lower esophageal sphincter, stomach, gallbladder), stimulate secretion of water into pancreatic juice and bile, and cause inhibition of gastric acid secretion and absorption from the intestinal lumen (Bowen et al.. “Vasoactive Intestinal Peptide,” Pathophysiology of the Endocrine System: Gastrointestinal Hormones. Colorado State University).
  • VIP stimulates intestinal secretion of water and electrolytes and relaxation of enteric smooth muscle, dilating peripheral blood vessels, stimulating pancreatic bicarbonate secretion, and inhibiting gastrin-stimulated gastric acid secretion (“Vasoactive intestinal polypeptide”. General Practice Notebook. Retrieved 2009-02-06). These effects work together to increase motility (Bergman et al., Atlas of Microscopic Anatomy: Section 6 - Nervous Tissue. URL :. anatomyatlases, org) .
  • VIP can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
  • Glicentin is a 69 amino acid peptide secreted after the processing of proglucagon along with the following peptides listed below:
  • Pre Proprotein Proglucagon Preproprotein
  • Signal peptide (1-20; SEQ ID NO:39) - removed from preproglucagon to form proglucagon
  • Glicentin-related pancreatic polypeptide (GRPP, 21-50; set forth in SEQ ID NO:25)
  • Oxyntomodulin (OXY or OXM, 53-89; set forth in SEQ ID NO:4)
  • Glicentin contains the entire sequence of glucagon and glicentin-related pancreatic polypeptide. There is no known glicentin receptor, so the mechanism of action is not well characterized. However, the concentration of glicentin in plasma is altered in patients experiencing diabetes and obesity, and following foregut surgery. Glucagon regulates systems metabolism including regulating acute and chronic thermogenic affects. (Kleinert et al., IntJMol Sci. 20(21): 5407 (2019))
  • Macrophage inhibitory cytokine 1 reduces body weight through appetite suppression.
  • Exogenous administration of recombinant GDF15 or GDF15 analogues decreases body weight in diet-induced obese mice and non-human primates (Mullican, S. E. et al., Nat. Med. 23, 1150-1157 (2017)).
  • GDF15 agents in phase 1 clinical studies include LA-GDF15 and the GDF15 agonists LY-3463251 and JNJ-9090/CIN-109.
  • any of the peptides listed above, and other peptides and therapeutics that are known or thought to impact (i.e., accelerate or initiate) weight loss or that are associated with a pathway involving hunger or digestion or metabolism can be administered in combinations and/or in accord with the rotational protocol set forth herein.
  • the peptides can be a synthesized peptide that can act as an agonist (or antagonist depending upon the pathway) for a receptor or receptors of any of the peptides and proteins and pathways detailed above, or pathways involving hunger, digestion and/or metabolism.
  • a single peptide in the combination and combinatorial rotational therapies provided herein can act as an agonist for multiple receptors of the peptides, proteins and/or pathways detailed above, or pathways involving hunger, digestion and/or metabolism.
  • a combination of any of peptides and/or other therapeutics administered in combination and/or in accord with a rotational regimen increases the efficacy of weight loss compared to administration of monotherapy or administration of a combination without the rotational component.
  • the protocols described herein and below are based on research that shows changes in hormonal secretions after bariatric surgery (see e.g., lonut et cd.. (2015) Obesity 21(6): 1093-1103).
  • the gut-brain axis is a major component of appetite regulation.
  • the gut hormones have either anorexic or orexigenic actions on food intake and it is now evident that these gut hormone secretions are altered following bariatric surgery.
  • regimens provided herein not only include combinations of therapeutics, but also can include a rotation of differing therapeutics, such as, for example, therapeutic peptides, such as rotating a peptide or combinations of peptides every 3 months.
  • the combination therapy can be rotated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 weeks or longer, such as every 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 months, or more.
  • the regimen can include rotation of different peptides every three months. This rotation will continue to drive weight loss as different receptors even among peptides within the same families will be activated.
  • the combination therapy described herein can be designed to mimic the hormonal and other signaling changes that occur after bariatric surgery, such as Roux-en-Y (Ochner etal., Int JObes 35(2): 153-166 (2011)).
  • the table below includes targets/pathways for targeting for weight loss, exemplary medications for targeting (z.e., activating or inhibiting) the pathways, the function of the administered medication, and exemplary references detailing the target pathways and/or medications for use for targeting the pathways.
  • Exemplary combinations for mimicking changes that occur following gastric bypass include exemplary medications for 2 or more of the targets listed in the table below:
  • Additional medications targeting pathways modified after bariatric weight loss surgery also can be added to the combinatorial regimen described herein.
  • Such medications include, for example, those that are not used because they are ineffective as a monotherapy, but as described herein can be effective, or efficacy can be increased compared to monotherapy, when administered in combination with other medications, for example in a combinatorial therapy, or a combinatorial rotational therapy.
  • ghrelin receptor antagonists medications that decrease circulating ghrelin, anti-ghrelin vaccines (e.g., the peptide vaccine CYT009-GhrQb), ghrelin receptor inverse agonists (GHSR-IA), the peptide-binding compounds (e.g., Nox-Bl 1), and any other medication(s) or therapy that decrease ghrelin or ghrelin- associated activation pathways can be included in the combination therapy herein.
  • receptor agonists that mimic the action of peptides that are increased after bariatric surgery can be included (z.e., PYY, CCK, and/or PP receptor agonists).
  • drugs for inclusion in the combination therapy herein also can include neuropeptide Y (NPY) antagonists, melanocortin-4 receptor (MC4R) agonists, cannabinoid-1 receptor antagonists, and agonists and antagonists as appropriate for increasing weight loss and/or decreasing the comorbidities of overweight and/or obesity.
  • NPY neuropeptide Y
  • M4R melanocortin-4 receptor
  • agonists and antagonists as appropriate for increasing weight loss and/or decreasing the comorbidities of overweight and/or obesity.
  • compositions provided for administration by a desired route, such as oral, mucosal, intravenous, and others.
  • Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans, and also, for agricultural applications, for plants.
  • compounds are formulated into pharmaceutical compositions using techniques and procedures well- known in the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).
  • oral drug delivery compared with injection or nasal spray administration are greater acceptability, ease of administration and convenience for patients.
  • oral delivery of a combination of medications i.e., therapeutic peptides
  • Oral delivery systems can pose a challenge due to proteolytic cleavage in the digestive tract, limited intestinal uptake of the intact molecule, and absorption of peptides that can be hindered by their physical characteristics, including the polarity and size of the dosage form, and their susceptibility to aggregation or degradation by the local pH in the tract.
  • most delivery vehicles cannot be administered orally, but some can be so-administered. Such vehicles can be selected if oral delivery is desired.
  • one or more of the following mechanisms can be utilized to enhance delivery of the combination of therapeutics described: 1) permeation enhancers with added glycosylation, PEGylation or lipidation to increase uptake across cell membranes; 2) enzyme inhibitors such as, for example, carnitine esters, soybean trypsin inhibitors, deoxycholic acids and organic acids to prevent degradation in the gut; 3) modulation of protein to prevent degradation, such as, for example, by adding stabilizing side chains to reduce degradation; 4) additional of particulate systems, as described herein, including polymeric micro and nanoparticles, liposomes, microemulsification or polymeric micelles; 5) addition of multifunctional polymers, to include, for example, Poly (alky cyanacryl ate) chitosan to assist with mucosal adhesion and bypass tight junctions; and
  • the therapeutic molecules in the combinations can be administered in forms that increase half-life.
  • the therapeutic molecules in the combinations can be provided as part of a liposome or multicellular laminar vesicle or other such delivery vehicle.
  • the therapeutic molecule provided in the combinations herein can be modified by a polymer prior to administration.
  • the polymer is a polyalkylene glycol, dextran, pullulan or cellulose.
  • Polyalkylene glycol polymers, which can modify the therapeutic molecule include polyethylene glycol (PEG) and methoxypolyethylene glycol (mPEG).
  • PEG polyethylene glycol
  • mPEG methoxypolyethylene glycol
  • the PEG can by branched or linear.
  • the polymer can be produced by reaction with methoxy -poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (5 kDa); methoxy-poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (20 kDa); methoxy-poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (30 kDa); methoxy -poly(ethylene glycol)- succinimidyl a-methylbutanoate (mPEG-SMB) (20 kDa); methoxy-poly(ethylene glycol)-succinimidyl a-methylbutanoate (mPEG-SMB) (30 kDa); methoxypolyethylene glycol)-butyraldehyde (mPEG-butyraldehyde) (30 kDa), m ethoxy - poly(ethylene glycol)-succin
  • any of the above approaches can be used to formulate the combination(s) of therapeutics described herein.
  • the combinations can be used for therapeutic, prophylactic, cosmetic, and/or diagnostic applications.
  • the combinations containing the therapeutics for the rotational combinatorial therapy described herein can be formulated with a pharmaceutical acceptable carrier or diluent.
  • such pharmaceutical combinations of compositions include components that do not significantly impair the biological properties or other properties of the cargo.
  • Each component in the combination is pharmaceutically and physiologically acceptable so that it is compatible with the other ingredients and not injurious to the subject to whom it is to be administered.
  • the formulations can be provided in unit dosage form and can be prepared by methods well-known in the art of pharmacy, including but not limited to, tablets, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, oil water emulsions, liquid solutions or suspensions (e.g., including injectable, ingestible and topical formulations (e.g., eye drops, gels, pastes, creams, or ointments)), aerosols (e.g., nasal sprays, and inhalers), liposomes, suppositories, pessaries, injectable and infusible solution and sustained release forms. See, e.g., Gilman, et al. (eds.
  • Each unit dose contains a predetermined quantity of therapeutically active compound or compounds sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit dose forms include ampoules.
  • the therapeutic composition When administered systemically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art. Methods for preparing parenterally administrable compositions are well-known or will be apparent to those skilled in the art and are described in more detail in, e.g., “ Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences)”, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
  • Pharmaceutical combinations provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, and lyophilized form.
  • suitable pharmaceutical carriers include but are not limited to water, buffering agents, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, and powders, among others.
  • compositions provided herein can contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacan
  • Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body.
  • the therapeutics for the rotational combinatorial therapy described herein can be prepared in a mixture with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Techniques for formulation and administration of the compounds are known to one of skill in the art (see e.g., “Remington's Pharmaceutical Sciences.'' Mack Publishing Co., Easton, Pa.).
  • This therapeutic composition can be administered intravenously or through the nose or lung, such as a liquid or powder aerosol (lyophilized).
  • the composition also can be administered parenterally or subcutaneously as desired.
  • the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
  • Therapeutic formulations can be administered in many conventional dosage formulations.
  • Dosage formulations of therapeutics for the rotational combinatorial therapy described herein can be prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers.
  • Such materials are non-toxic to the recipients at the dosages and concentrations employed, and can include buffers such as Tris HC1, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, di saccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN (polysorbates), Pluronic, polyethylene glycol, and others.
  • buffers such as Tris HC1, phosphate,
  • the formulation When used for in vivo administration (z.e., to patients), the formulation should be sterile and can be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • a therapeutic peptide for a rotational combination therapy described herein can be provided as lyophilized powder that is reconstituted with a suitable solution to generate a single dose solution for injection.
  • the therapeutics for the rotational combinatorial therapy described herein can be stored in lyophilized form or in solution; they can be frozen or refrigerated.
  • the lyophilized powder can contain the combination of therapeutics, such as a combination of therapeutic peptides, and additional components, such as salts, such that reconstitution with sterile distilled water results in a combination of therapeutics in a buffered or saline solution.
  • therapeutics such as a combination of therapeutic peptides
  • additional components such as salts, such that reconstitution with sterile distilled water results in a combination of therapeutics in a buffered or saline solution.
  • Unit dose forms can be administered in fractions or multiples thereof.
  • a multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials and syringes. Hence, multiple dose form is a multiple of unit doses that are not segregated in packaging.
  • the therapeutics for the rotational combinatorial therapy described herein can be provided at a concentration in the composition that is approved by regulatory agencies.
  • the pharmaceuticals are formulated and provided at a concentration for which the pharmaceuticals are approved (ie., as monotherapy or dual therapy).
  • the pharmaceutical composition contains multiple therapeutics in a combination and multiple different therapeutics for the rotational combinatorial therapy described herein.
  • the combination therapy described herein includes one or more additional agents, such as treatment of adverse side effects, or other therapeutic, for combination therapy.
  • compositions suitable for use include compositions wherein the therapeutics for the rotational combinatorial therapy described herein are contained in an amount effective to achieve their intended purpose.
  • previously FDA approved medication can be in an amount or dosage as previously approved or recommended by the FDA or any other regulatory agency.
  • the FDA approved medication used in a combination described herein can be provided in a lower amount or dosage than approved or recommended by the FDA or other regulatory agency.
  • multiple medications are provided in the combination in an amount or dosage that is approved or recommended by FDA.
  • multiple medications are provided in the combination in an amount or dosage that is less than the amount or dosage approved or recommended by FDA. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • Therapeutically effective dosages can be determined by using in vitro and in vivo methods, and/or by a skilled person.
  • Dosages generally start at FDA approved or recommended dosage. The lowest effective dosage can be indicated in order to decrease side effects. Dosages can be adjusted throughout the therapeutic period in accord with the regimen herein and also based on other factors. For example, dosage can be modified based on the particular subject’s response and/or progress (z.e., weight loss), tolerability, volume of distribution, subject weight and/or body size, and at the discretion of the treating physician. Metabolic parameters, cardiac markers for cardiac patients (z.e., CRP, ASR), inflammatory markers (z.e., myeloperoxidase (MPO)), renal clearance also can be assessed when determining dosage; dosages to decrease inflammation, decrease cardiac risk and that are renal protective would be preferred so long as the therapeutic efficacy is maintained.
  • CRP cardiac markers for cardiac patients
  • ASR inflammatory markers
  • MPO myeloperoxidase
  • any of the therapeutics for the rotational combinatorial therapy or combinations described herein can be formulated for single dosage or multiple dosage administration for use in the rotational therapy provided herein; generally, the therapeutics are formulated for multiple dosage.
  • an approved (z.e., FDA approved) therapeutic that is approved for single dosage administration is formulated for single dosage administration in the combinatorial therapy provided herein.
  • an approved (z.e., FDA approved) therapeutic that is approved for multiple dosage administration is formulated for multiple dosage administration in the combinatorial therapy provided herein.
  • a therapeutic that is approved for daily administration can be administered daily in the rotational combinatorial regimen described herein.
  • a therapeutic can be included in a first combination and rotated into the regimen as part of a later combination (z.e., fourth or fifth combination).
  • a therapeutic is provided in a combination and is not rotated into the regimen again; such as a therapeutic that is provided in a first combination only, and is not included in a later combination in the regimen.
  • the range of doses of any of the therapeutics in the provided combinations can be formulated per kg of body weight of the subject or patient.
  • the dose can be administered a single time during each rotation, or can be administered a multiple times.
  • Appropriate dose amount can be determined by one of skill in the art, based on the regimen of administration. Total dose over a specific period of time can also be selected by one of skill in the art.
  • the dose can be increased over time, during the regimen. In some examples the dose can be increased or decreased during the rotation, compared to the previously administered dose.
  • the dose range for each of the individual therapeutics in the combinations provided herein can be adjusted by plasma monitoring or by monitoring of symptoms or by monitoring of adverse side effects.
  • the dose of administration can be such that the subject will maintain a plasma level to effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered.
  • the dose of an individual therapeutic can vary depending on the other therapeutics in the combination, and can vary depending on the doses of the therapeutics in the other combinations in the rotation.
  • the therapeutics and combinations are included in amounts sufficient to exert a therapeutically useful effect in the absence of undesirable side effects, or with minimal or decreased side effects on the patient treated.
  • a therapeutically effective concentration of a therapeutic(s) for treatment of any condition with a previously characterized mechanism of action or pathway can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art and then extrapolated therefrom for dosages for humans.
  • the precise amount or dose of the therapeutic agent administered depends on the particular agent, the route of administration, the other agents in the combination, the other agents previously administered in the rotational regimen, the amounts (z.e., dosages) of the other agents previously administered in the rotational regimen, and other considerations, such as the severity of the disease and the weight and general state of the subject.
  • Local administration of the therapeutic agent will typically require a smaller dosage than any mode of systemic administration, although the local concentration of the therapeutic agent can, in some cases, be higher following local administration than can be achieved with safety upon systemic administration. If necessary, a particular dosage can be empirically determined or extrapolated.
  • the dosage of an approved (z.e., FDA approved) medication is the approved dosage or amount, or a standard dosage or amount, or a dosage or amount provided in applicable clinical guidelines (z.e., clinical guideline from the American Society for Radiation Oncology (ASTRO) provides guidance on the use of radiation therapy).
  • the dosage of a therapeutic in a combination is less than the dosage or amount approved by a regulatory agency.
  • the dosages of a therapeutic (z.e., medication) in a combination can be decreased compared to the dosage of the therapeutic when administered as a monotherapy.
  • the amount of any therapeutic in the combinations provided herein to be administered for the treatment of a disease or condition can be determined by standard clinical techniques.
  • in vitro assays and animal models can be employed to help identify dosage ranges for administration.
  • animal models can be used to establish an effective dosage.
  • One of skill in the art can determine the human equivalent dose (HED), based body weight for humans. Appropriate HED can be calculated using body weight-based conversion (Reagan-Shaw et al. (2008) The FASEB Journal 22(3):659-661).
  • the precise dosage which can be determined empirically or based on previous use of the therapeutic (z.e., the standard dosage), can depend on the particular agent, the route of administration, the type of disease to be treated, the particular disease or condition, the particular formulation, the seriousness of the disease or condition, and other factors within the level of a skilled artisan.
  • the combinations administered can contain therapeutic peptides that mimic the peptides released or increased following bariatric weight loss surgery provided herein, such as, for example, gastrointestinal hormones, PYY and/or GLP-1.
  • the therapeutic peptides are administered in a dosage that effects levels (z.e., circulating levels) of the peptides similar to the levels following bariatric weight loss surgery.
  • the dosage of an individual therapeutic in the combination is the dosage approved by a regulatory agency for administering an approved therapeutic as a monotherapy.
  • dosage and duration of treatment is a function of the disease being treated and the dosage and duration of treatment can follow a previously established schedule for administration of an approved therapeutic (z.e., drug).
  • Therapeutic dosage and duration of treatment also can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.
  • One of skill in the art can, in a clinical setting, and in accord with the rotational methods and combinations described herein, determine the dosage of individual therapeutics in the combinations provided herein for administration. Dosage and duration of treatment also can be modified considering the other therapeutics in the combination, and in the rotational therapy. Concentrations and dosage values also can vary with the severity of the condition to be alleviated. The dosages can depend on the clinical response and side effect profile of an individual subject, such as a human or an animal.
  • dosages and dosage regimens for the combination therapy are chosen to limit toxicity and/or adverse side effects.
  • the therapeutic can initially be administered at a low dosage and increased gradually according to patient's response.
  • the therapeutic can initially be administered at the standard dosage and can be increased gradually according to the patient’s response.
  • the combinations can be administered hourly, daily, bi-weekly, weekly, monthly, once, twice daily, three times a day, four times a day, five times a day, or more.
  • different therapeutics in a combination are administered with the same frequency.
  • three therapeutics in a first combination are administered once daily.
  • four therapeutics in a first combination are administered twice daily.
  • the frequency of administration can vary as long as all of the therapeutics are administered with the same frequency (z.e., at the same time).
  • therapeutics in a combination are administered on distinct schedules (z.e., with different frequency).
  • one therapeutic in a first combination is administered twice daily, and a second therapeutic in the first combination is administered once daily, and a third therapeutic in the first combination is administered bi-weekly.
  • the therapeutics are administered in accord with their approved administration monotherapy timing.
  • therapeutics that are administered daily as a monotherapy are administered daily in the combination regimen provided herein.
  • therapeutics that are administered weekly as a monotherapy are administered weekly in the combination regimen provided herein.
  • therapeutics that are administered biweekly as a monotherapy are administered bi-weekly in the combination regimen provided herein.
  • the delivery vehicles such as the liposomes, provided herein can be formulated for administration by any suitable route, including oral and injection, such as subcutaneous injection.
  • liposomes displaying combinations of peptides as exemplified can be formulated for subcutaneous administration, particularly for self-administration.
  • Numerous injectors, autoinjectors, and high speed autoinjectors are known in the art see, e.g., U.S. Patent Nos.
  • An appropriate single dose or multiple dose can be loaded into an injector for administration.
  • the liposomes display a plurality of peptides (or also contain a small molecule)
  • the dosage amounts of the liposomes can be provided in the injector for use by a subject.
  • a typical volume is 1 mL to 3 mL (or a range therein) or up to about 5 mL, and in some instances, where the small molecule includes about 10 mL.
  • the liposomes can be produced that display 1 to 3 peptides.
  • the liposomes can be separately administered or mixed and administered in a single composition, which can be loaded into an injector, such as for subcutaneous administration. Liposomes, such as those exemplified herein, can be administered via injection.
  • the small molecules, such as phentermine can be administered as a separate dosage form, such as a tablet or capsule. They can be administered together, or sequentially or intermittently with the delivery vehicles. Generally, they can be included in or on the delivery vehicles.
  • the combinations can be formulated using previously approved (ie., approved by the US Food and Drug Administration) dosages.
  • GLP-1 agonists can be formulated at the starting dosage in the combinations that is the starting dosage of the GLP-1 agonist administered as a monotherapy.
  • dulaglutide (Trulicity®) GLP-1 agonist can be formulated in the combination at a starting dosage of 0.75 mg administered once weekly
  • GLP-1 agonist can be formulated in the combination at a starting dosage of 2 mg administered once weekly
  • semaglutide (soled as Ozempic®) GLP-1 agonist can be formulated in the combination at a starting dosage of 0.5 mg administered once weekly.
  • Exemplary medications and their starting dosages are set forth in Table 11, below.
  • combinations for rotational combinatorial therapy for treating obesity or overweight can be formulated and administered in the therapeutic amounts or ranges set forth in Table 12, below:
  • a pharmaceutical combination for treating overweight, obesity and/or for weight loss including FDA approved medications.
  • one or more of the following is included the combination: 1) a GLP-1 agonist (sold under trademarks Trulicity®, B-cise®, Ozempic®, and Victoza®), 2) Phentermine, 3) Liothyronine, 4) topiramate (sold under the trademark Topamax® carbonic anhydrase inhibitor), 5) Sermorelin, and 6) tirzepatide (sold under the trademark MounjaroTM).
  • one or more of the following is administered: one or more GLP-1 agonists, such as, for example Trulicity®, BYDUREON Bcise®, Ozempic®, Victoza® GLP-1 agonist; Phentermine; Liothyronine; topiramate (such as Topamax® carbonic anhydrase inhibitor); Acarbose; Sitagliptin (Januvia® dipeptidyl peptidase-4 (DPP-4) inhibitor); Canagliflozin (Invokana® sodium-glucose co-transporter 2 (SGLT2) inhibitor); Dapagliflozin (Farxiga® SGLT2 inhibitor); tirzepatide; and Sermorelin.
  • GLP-1 agonists such as, for example Trulicity®, BYDUREON Bcise®, Ozempic®, Victoza® GLP-1 agonist
  • Phentermine Liothyronine
  • topiramate such as Topamax® carbon
  • one or more of the medications in the pharmaceutical combination is discontinued and restarted.
  • one medication z.e., phentermine
  • another medication z.e., Liothyronine
  • a medication or medications is started after the first medication(s) is/are administered (the regimen is started), for example 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more after the regimen is started.
  • the methods and regimens provided herein include a method of treating an obese subject with a rotational combination therapy comprising administering a plurality of combinations of therapeutics to the subject.
  • An effective amount of the therapeutics in the combinations readily is determined by one of skill in the art to effect weight loss in the subject. Doses of the therapeutics in the combinations or the timing of the treatment regimen can be varied or adjusted based on the susceptibility of the patient to the treatment, as determined by one of skill in the art, such as by using the methods provided herein.
  • dosages can be provided as a ratio of the amount of one of the therapeutics in the combination to the amount of the other therapeutic agent(s) administered.
  • the therapeutics in the combination therapy and the combinations provided herein can be formulated for any route known to those of skill in the art including, but not limited to, subcutaneous, intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, epidural, vaginal, rectal, local, optic, transdermal administration, or any route of administration.
  • subcutaneous administration the single dosage is administered in volume that is generally 10 ml or less, such as 1-5 ml, 1-2 ml, 2-10 ml, no more than 4, 5, 6, 7, 8, or 9 ml.
  • Formulations suited for such routes are known to one of skill in the art.
  • Formulations of previously characterized (z.e., FDA approved) therapeutics can be administered in the combinations herein in accord with the previously characterized administration route.
  • Therapeutic compositions also can be administered concomitantly with other therapeutics in the combination.
  • Therapeutic compositions also can be administered with other biologically active agents including therapeutics in the combination or other biologically active agents, either sequentially, intermittently or in the same composition.
  • the combination therapy can be administered by any suitable route, and for any use for which the combination of therapeutics are used, including treatment of obesity or overweight.
  • compositions can be administered by controlled release formulations and/or delivery devices (see, e.g., in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,660; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,556; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).
  • Various delivery systems are known and can be used to administer selected compositions, are contemplated for use herein, and such particles can be easily made.
  • the route of administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical, rectal, mucosal, and by sustained release systems.
  • the combinations containing the therapeutics for the rotational combinatorial therapy described herein can be administered continuously by infusion or by bolus injection.
  • the skilled physician can administer the combinations containing the therapeutics for the rotational combinatorial therapy described herein in a local or systemic manner.
  • Described herein is an improved pharmacotherapeutic cocktail that mimics bariatric weight loss surgery and includes combining the selected peptides at the right physiologic time (i.e., prior to meals, or early or later in the day) and developing stable and soluble peptides that will be easily administered through subcutaneous injection, or intramuscular injection, or by oral formulation.
  • Each of the peptides described herein form a part of the message which becomes integrated at different levels of the brain and produces an outcome of either feed or do not feed.
  • Table 13 sets forth an exemplary protocol for administering a combinatorial therapy, including 6 different combinations, each comprising more than one therapeutics, where the combinations are rotated after 4 months of administration.
  • the therapeutics in the combinations target different pathways, for different effects.
  • compositions containing the combinations for the rotational therapy described herein can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a disease or condition that can be treated by rotational administration of the particular combinations, such as the diseases and conditions described herein or known in the art, and a label that indicates that the cargo, such as a drug or other therapeutic (z.e., a weight loss medication), is to be used for treating the condition, disease or disorder.
  • the pharmaceutical compositions can be packaged in unit dosage forms containing an amount of the pharmaceutical composition for a single dose or multiple doses.
  • the packaged compositions can contain a lyophilized powder of the pharmaceutical compositions containing the combinations for the rotational therapy which can be reconstituted (e.g., with water or saline) prior to administration in accord with the rotational regimen.
  • the articles of manufacture provided herein contain packaging materials.
  • Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252).
  • Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • MDI pressurized metered dose inhalers
  • DPI dry powder inhalers
  • nebulizers e.g., jet or ultrasonic nebulizers
  • the combinations provided herein can be packaged in accord with the packaging of the individual medications as
  • kits optionally can include one or more components such as instructions for use, devices, and additional reagents (e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods.
  • Exemplary kits can include the therapeutic(s) in the combinations for rotational therapy provided herein, and can optionally include instructions for use, a device, such as a syringe or other injector, for administering the combinations for rotational therapy to a subject, and a device for administering an additional therapeutic(s) agent to a subject.
  • the kit can, optionally, include instructions. Instructions typically include a tangible expression describing the combinations for rotational therapy, and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the combinations for rotational therapy. Instructions also can include guidance for monitoring the subject over the duration of the treatment time. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount, or activity of therapeutic(s) in the combination for rotational therapy, in a subject.
  • Kits provided herein also can include a device for administering the combinations to a subject. Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a nebulizer, and an inhaler. Typically, the device for administering the compositions is compatible with the desired method of administration.
  • the delivery vehicles such as the liposomes herein are known.
  • the delivery vehicles can be administered by injection, such as subcutaneous or intramuscular injection.
  • the peptide drugs are known and can be delivered by subcutaneous injection, and are commercially available in autoinjectors or other such devices that that deliver predetermined amounts of a product.
  • the product can be provided in the device as a liquid, or the device can contain chambers in which a lyophilized product is separate from a liquid vehicle, such as PBS, for administration. A membrane separating the chambers can be pierced before using and the lyophilized product dissolved in the vehicle.
  • the injectors can include multiple chambers, if the drugs are provided, on different liposomes. Appropriate amounts to achieve a desired dosage of each are mixed before injection.
  • Autoinjectors that contain a fixed dosage amount or multiple dosages of the delivery vehicles.
  • Autoinjectors include spring-actuated mechanical devices, which are user administered by pressing a button or pushing against an injection site (push-on-skin), which provides a pre-determined fixed volume from a pre-filled syringe.
  • Autoinjectors can include visual and audible cues, and, in general, permit non-healthcare professionals to administer medications safely and effectively.
  • Autoinjectors for use in the delivery drugs including biologies and small molecules are well known and readily accessible to the public.
  • Examples of approved drugs administered by autoinjectors include, but are not limited to, Epinephrine (Adrenaline) (e.g., EpiPen® autoinjector system), Naloxone (e.g., EZVIOTM naloxone autoinjector), Atropine (and Pralidoxime), Diazepam, Sumatriptan (e.g., Alsuma/StatDose), Abatacept (e.g., ORENCIA® ClickJectTM single-dose prefilled autoinjector), Etanercept (e.g., lyophilized Enbrel® reformulated for the SureClick® autoinjector), Mepolizumab (e.g., NUCALA® autoinjector), and Benralizumab (e.g., Fasenra® autoinjector), and commercially available versions of the weight loss peptides, such as the GLP-1 receptor agonists and others.
  • Epinephrine Adrenaline
  • Autoinjectors are an be used in the delivery of fixed volumes of injectable fluid, where the injectable fluid contains the delivery vehicles or the delivery vehicles and any suitable excipient.
  • Exemplary autoinjector systems are described in U.S. Pat. Nos. 7,618,396, 7,731,686, 8,021,335, 8,647,299, 8,945,067, 9,333,304, 9,408,973, 9,421,337, 10,493,212, 10,695,492, 10,716,901, 11,058,820, 11,167,087, 11,285,266, 11,648,355, 11,992,663 and 12,005,236, 12,208,245, and U.S. 2024/0207375
  • U.S. patent publication 2024/0207375 describes high volume autoinjectors for use for subcutaneous administration.
  • Exemplary routes of administration for auto injection include subcutaneous and intramuscular delivery.
  • Exemplary FDA and EMA approved subcutaneous syringes, prefilled needle safety devices, and prefilled handheld autoinjectors include, for example:
  • Handheld autoinjectors optimized for doses less than about 2.0 mL e.g.,
  • autoinjectors capable of delivering higher doses, ranging between 2 and 5 mL or more (e.g., YpsoMate® 5.5) also are available.
  • the single dosage is administered in volume that is generally up to 10 mL, or about 10 ml or less, such as 1-5 ml, 1-2 ml, 1-3 ml, 2-10 ml or other such amounts.
  • Auto injection rates range from at least or about 0.003 mL/s to about 1.0 mL/s.
  • a high volume autoinjector can be configured to subcutaneously administer a formulation to a subject at a rate of about 0.05 mL/sec to about 1.0 mL/sec.
  • the speed at which a high volume of the formulation can be administered to the subject depends on the gauge of the needle used to inject the formulation.
  • the size and injection rate of the autoinjector can be configured to meet the needs of the end-user, see, e.g., U.S. 2024/0207375.
  • Exemplary commercial handheld autoinjectors at or exceeding 3.0 mL for subcutaneous administration include:
  • Wearable devices or on-body delivery systems provide for subcutaneous delivery of volumes greater than 3mL.
  • Exemplary devices include SmartDose® 3.5 injector (West Pharma, Exton, PA, USA), CRONO ambulatory infusion pumps (Cane Medical Technology, Rivoli TO, Italy), Infusion Pump (Sensile Medical, Olten, Switzerland), LibertasTM autoinjector (BD, Franklin Lakes, NJ, USA), Lapas® patch pump (Bespak, King’s Lynn, UK), enFuse® on-body platform (Enable Injections, Cincinnati, OH, USA), Wearable injectors (Sonceboz, Sonceboz- Sombeval, Switzerland), Wearable injection devices (Sorrel Medical, Netanya, Israel), DrugDeliverySystems (Weibel, Switzerland), and YpsoDose® (Ypsomed, Burgdorf, Switzerland).
  • Auto injection rates range from at least or about 0.003 mL/s to about 0.1 mL/s, and at least or about 0.1 mL/s to about 0.9 mL/s; higher speed injectors also are available.
  • auto injection volumes range from at least or about 0.1 to about 0.5 mL, at least or about 0.1 to about 1.0 mL, at least or about 0.1 to about 1.5 mL, at least or about 0.1 to about 2.0 mL, at least or about 0.1 to about 2.5 mL, at least or about 0.1 to about 3.0 mL, at least or about 0.1 to about 3.5 mL, at least or about 0.1 to about 4.0 mL, at least or about 0.1 to about 4.5 mL, at least or about 0.1 to about 5.0 mL, at least or about 0.1 to about 5.5 mL, at least or about 0.1 to about 6.0 mL, at least or about 0.1 to about 6.5 mL, at least or about 0.1 to about 7.0 mL, at least or about 0.1 to about 7.5 mL, at least or about 0.1 to about 8.0 mL, at least or about 0.1 to about 8.5 mL, at least or about 0.1 to about 9.0 m
  • Excipients that facilitate subcutaneous injection can be included in the formulations and pharmaceutical compositions.
  • the delivery vehicles can be coformulated with such excipient, such as with a hyaluronidase, such as a soluble human hyaluronidases, such as the commercially available ENHANZE® soluble human hyaluronidase.
  • a hyaluronidase such as a soluble human hyaluronidases, such as the commercially available ENHANZE® soluble human hyaluronidase.
  • a hyaluronidase in a formulation, such as those provided herein, reduces dose administration time, dosing frequency, and provides for the delivery of large volumes for rapid subcutaneous injections, such as 5 to 15 mL, and infusions, up to 600 mL.
  • compositions that contain the delivery vehicles and an excipient, such as a hyaluronidase, such as a human soluble hyaluronidase
  • Pharmacokinetic and tolerability studies can be performed using animal models or can be performed during clinical studies with patients to assess the effect of the combinations provided herein.
  • Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques.
  • pharmacokinetic and tolerability studies are performed using healthy animals.
  • the studies are performed using animal models of a disease for which therapy with a combination herein is considered, such as animal models of obesity, or animals with metabolic disorders (ie., transgenic animals that model or show characteristics of metabolic disorders).
  • animal models of diabetes e.g., diabetes mellitus type 2
  • cardiovascular disease z.e., cardiovascular disease leading to heart attack or stroke
  • high blood pressure high blood cholesterol
  • high triglyceride levels persisting neurodegenerative disorders, such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and/or nonalcoholic fatty liver disease
  • neurodegenerative disorders such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and/or nonalcoholic fatty liver disease
  • the pharmacokinetic properties and activities of therapeutics in the rotational combinatorial therapy herein can be assessed by direct and indirect methods.
  • the concentration of the therapeutics (z.e., drug) such as, for example, in the blood or serum or other body fluid
  • the concentration of the therapeutics can be assessed after therapeutic administration or at a time point following administration, such as, for example, at or about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks or more after administration of the rotational combinatorial therapy.
  • the concentration of the active drug ingredient in the blood can be assessed.
  • the equation of bioavailability can be used for assessing the therapeutic effect.
  • Indirect methods also can be used to assess the pharmacokinetic properties and activities of therapeutics, and the therapeutic effect(s).
  • the rate of urinary excretion such as cumulative urinary excretion
  • the drug pharmacokinetics can be assessed by evaluating drug absorption, distribution, bioavailability, metabolism and elimination.
  • a range of doses and different dosing frequency of dosing can be administered in the pharmacokinetic studies to assess the effect of increasing or decreasing concentrations of the therapeutics in the combinations.
  • Pharmacokinetic properties, such as bioavailability, of individual drugs in the combination also can be assessed with co-administration of other therapeutic(s) in the combination.
  • animal models can be administered the therapeutics in a first combination, using one or more routes of administration. Such studies can be performed to assess the effect of co-administration of the plurality of therapeutics (z.e., drugs) in the combination.
  • Adverse reactions can include, but are not limited to, injection site reactions, such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses.
  • injection site reactions such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses.
  • a range of doses and different dosing frequencies are be administered in the safety and tolerability studies to assess the effect of increasing or decreasing concentrations of the active agent.
  • Peptides can be displayed or provided in delivery vehicles, such as liposomes. Small molecule drugs and larger polypeptides cand be provided in delivery vehicles, such as liposomes, as described in the Examples, or they can be provided separately for administration, generally for separate administration.
  • the peptides can be modified to include a K (Lys) near or at the C-terminus for pegylation, such as to link to the liposomes as exemplified and described herein.
  • the position of the K is selected so that, when displayed, the peptide retains activity.
  • the peptides also can be modified to replace other K residues with a conservative amino acids, such as R, so that only the K near the C-terminus (or a locus that does not affect activity) is pegylated.
  • Peptides and proteins typically contain many amino groups. At least one is terminal, and there might be several lysine side chains, each having one amino group. These can be functionalized with carboxylic acid active ester, such as NHS ester. Alternatively, NHS ester can be created in situ.
  • 1 pmol protein or peptide is dissolved into 10 ml PBS.
  • 10 pmol polyethylene glycol diacid is added (1ml 10 mM solution).
  • 10 pmol NHS is added (1ml 10 mM solution, and 1ml 1 mM solution, respectively).
  • EDC N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
  • 10 min 1ml more 1 mM solution of EDC is added.
  • the recti on mixture is transferred into 100 ml Sephadex G-100 column. The column is eluted with Tris buffer that is pumped with peristaltic pump 5 ml/min.
  • PEG-peptide After about 30 min the PEG-peptide has peak value. Fractions (0.5 ml) are collected into microcentrifuge tubes. 10 pl samples are diluted into 1 ml, and absorbance is measured at 260 nm. PEG-peptide is eluted first, then unreacted peptide, and finally unreacted PEG diacid. PEG-peptide fractions are combined and used in Example 3. This reaction is repeated for each protein or peptide separately. These PEGs are carboxylate terminated. Biotin terminated PEG-peptides are obtained by using biotin-PEG carboxylate as a starting material.
  • the peptide solutions/suspensions can be lyophilized use, such as in Example 3.
  • the PEG moiety is 24 units long. Any size PEG can be employed. For example, longer PEG moieties can be employed. If needed, click chemistry can be used to extend the PEG spacer.
  • 1 g of t-BOC-lysine (Sigma- Aldrich) is dissolved into acetonitrile/methanol 20: 1. Dicyclohexyl carbodiimide is added. After 1 h, the mixture is filtered to remove dicyclohexyl urea, and the filtrate is concentrated in a rotary evaporator to 10 ml. The product is purified using HPLC. The purified product is depicted in Figure 5C.
  • Azido-dPEG24-acid is mixed with 25 mg (0.1 mmol) t- BOC-lysine methyl ester in 10 ml of acetonitrile. 21 mg (0.1 mmol) Dicyclohexyl carbodiimide is added. After 1 h the mixture is filtered, and 1 g of trifluoroacetic acid is added. After 30 min the mixture is purified with HPLC.
  • the purified product can be used in peptide synthesis to produce peptides with one or more Pegylated lysines.
  • the PEG moieties are for linkage to the liposomes.
  • the purified PEGylated peptide product is depicted in Figure 5B. Generally, the most C-terminal lysine is pegylated. As needed the lysine for pegylation is one that will not eliminate activity.
  • the peptides can be produced by other methods, such as by recombinant expression in a suitable host, such as E. coli. Lysines for which pegylation could affect or eliminate activity can be replaced with a conservative amino acid replacement, such as Arg, Glu, Gin, His).
  • Liposomes from Example 1 and applicable PEG-peptides from Example 2 are combined. 10 pmol NHS, and 1 pmol N-(3-dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (EDC) is added (1ml 10 mM solution, and 1ml 1 mM solution, respectively). pH is adjusted to 9 using sodium carbonate. After 20 min 1ml more 1 mM solution of EDC is added. After 1 hour the rection mixture is transferred into 100 ml Sephadex G-100 column. The column is eluted with Tris buffer that is pumped with peristaltic pump 5 ml/min. After about 20 min the peptide-liposomes have the peak value.
  • EDC N-(3-dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride
  • Liposomes are prepared as in Example 1, except, 5 mg of PE is used 2 mg of biotin-PEG-NHS (Sigma-Aldrich) is added, and pH adjusted to 9 using sodium carbonate. After 1 hour the mixture is dialyzed against PBS. After 6 hours streptavidin in PBS added (same amount as lipid in liposomes). Excess streptavidin is removed by Sephadex G-100 column.
  • the peptides are functionalized similarly with biotin-PEG-NHS, and also dialyzed. These biotin-PEG-peptides are added into liposome solution, and will bind spontaneously. Unbound peptides can be removed by a Sephadex column.
  • biotin terminated oligonucleotide can be bound to the streptavidin liposomes.
  • the peptides can be conjugated to biotin terminated oligonucleotides that are complementary to those on the liposomes.
  • Fig. 1 schematically depicts a short segment of a polypeptide.
  • Amino acids k, 1, and m are lysines, and can be separated from each other by several amino acids.
  • carboxylic group is protected by a methyl group that is removed by basic hydrolysis before the next step.
  • the methyl ester of next amino acid is added together with a condensing agent that often is dicyclohexyl carbodiimide (DCCI).
  • DCCI dicyclohexyl carbodiimide
  • the polypeptide is often synthesized on a solid phase surface onto which the growing peptide chain is chemically attached. This allows the washing of the unused reagents and soluble reaction products off.
  • the epsilon amino group When lysine is added into a polypeptide chain, the epsilon amino group must be protected. Possible protective groups are allyloxycarbonyl and trityl groups. These are orthogonal to other protective groups. In some instances, the Epsilon-amino group of lysine can be reacted with carboxyl terminated PEG. The bonding will be an amide bond that renders the amino group totally inert. The other advantage is that the peptide contains a PEG in a desired site. The other end of PEG should have a functional group that can be used for the conjugation with a liposome.
  • the functional group can be acetylene that can be reacted with azide that is in the liposome (click chemistry), or alternatively azide group, and the liposome has an acetylene.
  • the other lysines can have conventional protective groups, unless several PEGs are intended for the polypeptide. No separate PEGylation step is needed, since the polypeptide as synthesized is PEGylated. The peptide is synthesized in PEGylated form.
  • Fig.2 shows the product, in which lysine 1 is PEGylated, and PEG has an azide group at the end.
  • Azide as exemplified below, can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry).
  • PEG-lysines can be added either in amino or carboxylic end of the polypeptide. This kind of addition can be done anyway in order to make the binding of the polypeptide with liposome stronger.
  • the PEG is 24 units long. Longer PEG moieties can be used, and/or click chemistry, such as discussed below, can be used to extend the PEG spacer.
  • Liposomes can be fabricated in many ways, including heating methods, membrane contractor, and vertic flow focusing (VFF). See, e.g., the following references for each method: (1) Heating method: Mozafari M.R., Reed C.J., and Rostron C., 2002. Development of nontoxic liposomal formulations for gene and drug delivery to the lung. Technology and health care, 10 342- 344; (2) Membrane contractor: Jaafar- Maleej, C., Charcosset, C., and Fessi, H., 2011. A new method for liposome preparation using membrane contractor.
  • VFF vertic flow focusing
  • VFF Vertical Flow Focusing
  • Another advantageous method is high pressure homogenization, which is readily scaled up to industrial production, and produces small (100 nm) liposomes of uniform size and compositions; and high concentrations (up to 15% by weight) can be achieved.
  • Liposomes that contain a DBCO group can be prepared.
  • the DBCO group reacts with azido-tagged peptides.
  • PEG-phospholipids are commercially available, such as, from Pure Peg (see, biochempeg.com).
  • One end of the PEG is attached to a phospholipid, and the other end of the PEG has a dibenzocyclooctyne (DBCO) group for click chemistry to link to the peptides.
  • DBCO dibenzocyclooctyne
  • DBCO reagent is a well-known class of click chemistry labeling reagents that react with azide-tagged molecules. DBCO groups can exclusively react with azide- tagged molecules. 2K, 3.4K, and 5K, corresponding to molecular weights of 2.9 kD, 4.3 kD, and 5.9 kD, respectively, of these compounds are available. Equal molar amounts of each is used. For example, if 1 mmole (760 mg) of phosphatidyl choline is used, the total amount of DBCO phospholipid is 0.12 mmole (i.e. 0.04 mmole of each. 0.01 mmole is 29 mg (2K), 43 mg (3.4K), and 59 mg (5K). 0.04 mmoles is 116 mg, 172 mg, and 236 mg, respectively.
  • the total weight of all lipid components is 1274 mg. 30 ml of buffer is used to disperse this amount as liposomes. The minimum volume to disperse this amount of liposomes is about 10 ml.
  • lOx For coupling of peptides, lOx more of each peptide than used in the following example (or other peptides therapeutics, including any described herein) of peptide fabrication can be used. Azidopeptides couple spontaneously with DBCO liposomes.
  • 3 ml of the resulting compositions contains as much of each peptide as occurs in blood. Smaller amounts, however, can be used for administration. Thus, for example, about 1 ml to 3 ml of the liposomes displaying the peptides can be used.
  • Azido-dPEG24-acid is mixed with 25 mg (0.1 mmol) t- BOC-lysine methyl ester in 10 ml of acetonitrile. 21 mg (0.1 mmol) Dicyclohexyl carbodiimide is added. After 1 h the mixture is filtered, and 1 g of trifluoroacetic acid is added. After 30 min the mixture is purified with HPLC. The purified product (Fig. 3) can be used in the peptide synthesis.
  • Cholesterol-PEG-DBCO constructs are readily available from, for example, creativepegworks.com, MW 2k, 3.4k, 5k, 10k, and Ik. These constructs react with azide in copper free click chemistry.
  • Cholesterol MW is about 400g/mol.
  • Liposomes can contain 40 mol % of cholesterol. 1/40 of this can be PEG-DBC0 derivatives. Out of 100 mg cholesterol, 75 mg actual cholesterol, 30 mg C-PEG-DBCO MW 2k, 48 mg C-PEG-DBCO MW 3.4k, 150 mg C-PEG-DBCO MW 10k.
  • Liposomes should contain 89 % egg PC, and 11 % palmitoyl oleoyl PE of total phospholipids.
  • the resulting liposomes contain 156 mg cholesterol, 2.5 mg MW 2k, 4 mg MW 2k, 6.4 mg MW 3.4k, 21 mg MW 10k of the constructs.
  • Peptide 1 GLP-1 1.68 ug 100 pM
  • Peptide4 Oxyntomodulin 4.45 ug 200 pM
  • Peptide5 Sermorelin 300 ug 30 ug/kg
  • PeptidelO Enterostatin/GIP 15 mg 600 nM
  • Peptide4 Oxyntomodulin 45 ug 10 nmol
  • Peptide5 Sermorelin 3 mg 1 umol
  • Total amount of phospholipids in the liposomes is 1.12 mmoles in this example.
  • the amount of PEG spacers is 5 pmoles.
  • the amount of peptides can be slightly less than 5 pmoles. See Example above for fabrication of liposomes that can contain 10-fold more peptide.
  • Liposomes can carry drugs in many ways. On the outer surface can be chemically bound peptides or other big molecules using spacers, on outer and inner surface molecules can be adsorbed by ionic bonds, bilayer can incorporate lipid soluble drugs, such as steroid hormones, for example testosterone or estrogen, and inner space can contain water soluble drugs.
  • lipid soluble drugs such as steroid hormones, for example testosterone or estrogen
  • inner space can contain water soluble drugs.
  • phentermine hydrochloride is a known weight loss drug. Using phentermine hydrochloride has an added advantage that it is released slowly after the peptides have been inactivated by proteases. Slow release also suppresses possible side effects that might be observed, if phentermine is digested as a tablet.
  • liposomes and peptides are fabricated separately.
  • the small molecule such as phentermine hydrochloride
  • Phentermine is not removed from outside of the liposomes after the fabrication. Once fabricated they can be mixed in the quantities indicated above. They couple spontaneously without any catalyst.
  • the volume of a liposome can be 500 000 nm 3 1 mg of water is 10 18 nm 3 .
  • water in one liposome weighs 5 x 10' 13 mg.
  • 10 14 liposomes have 50 mg of water, and 10 16 liposomes have 5g of water inside. 5g of water is able to dissolve 10 mg of phentermine hydrochloride. If the liposomes are tightly packed, their total volume including water inside and outside would be 10 ml. This would still be reasonable volume for one dose. This is maximally tight packing.
  • a recommended volume for 10 16 liposomes is 1000 ml or one liter But the dosage can be lower, and liposomes modified to increase the amount of phentermine.
  • 10 16 liposomes have 2 x 10' 3 moles or 2 mmoles of phospholipids, i.e., about 1.4 g. If 10 % of total phospholipids are acidic, such as phosphatidic acid or phosphatidyl glycerol, there would be 0.2 mmoles of acidic phospholipids. Each acidic phospholipid molecule can bind one phentermine molecule. Molecular weight of phentermine is 150 g/mol, and 30 mg of phentermine would be bound. Half of that is outside, and would be released fast. The other half will be released slowly.
  • the amount used can be substantially lower, since a single dose of phentermine, since a dose of phentermine, when used alone, is about 15 mg to 37.5 mg. In the combinations herein, the dose can be substantially lower. Because phentermine is combined with other drugs, the dosing is lower 10 ml (about 5 mg of phentermine) or significantly less, so that dosage of the resulting liposomes is about 1 ml to 5 ml.
  • examples of combinations of polypeptides for displaying on or delivering in a delivery vehicle include combinations of at least two or three weight loss drugs and a muscle enhancer, and can further include a small drug, such as an appetite suppressor, an amphetamine family drug, and/or drugs for treating obesity co-morbidities.
  • the small molecules can be fabricated in association with the delivery vehicle, such as the liposomes or exosomes, or can be separately administered. Examples of fabrication of liposomes is detailed in the above examples. Other methods for synthesizing liposomes or other delivery vehicles, such as exosomes, are known and/or apparent to those skill in the art.
  • Drug 1 GLPl/GIPl/Oxyntomodulin + ME* (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 4 + SEQ ID Nos: 5, 8, 44
  • Drug 2 GLPl/GIPl/Amylin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 7 + SEQ ID Nos: 5, 8, 44
  • Drug 3 GLP1/GIP1 /Glucagon + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 27 + SEQ ID Nos: 5, 8, 44 Drug 4: GLP1/GIP1/CCK+ ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 11 + SEQ ID Nos: 5, 8, 44
  • Drug 5 GLP1/GIP1/PYY + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 6 + SEQ ID Nos: 5, 8, 44
  • Drug 6 GLP1/GIP1 /Leptin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 3 + SEQ ID Nos: 5, 8, 44
  • *ME A muscle enhancer such as Sermorelin or Tesamorelin or IGF1 or other muscle enhancing peptide or drug that ameliorates the loss of muscle accompanying weight loss by promoting muscle growth
  • the peptides are provided on delivery vehicles, such as liposomes and exosomes or other lipid particles.
  • the delivery vehicles such as liposomes, display at least two peptides for weight loss, and at least one for muscle enhancement for administration.
  • delivery vehicles displaying a single peptide are prepared, and combinations thereof, each displaying one peptide are administered together in separate compositions or in co-formulations.
  • the peptides and regimens can be designed in accord with the Examples below and description herein.
  • the compositions can be provided in pens for injection.
  • the compositions can be formulated for other routes of administration, such as mucosal and, for some vehicles, oral, depending upon the particular delivery vehicle.
  • This Example provides weight loss and other data achieved with existing weight loss drugs/medications and protocols for comparison with the results achieved using the combinatorial method provided herein in which combinations of drugs are administered, and in which the combinations can be rotated.
  • gastric bypass is the most effective weight loss protocol. It, however, is fraught with adverse side-effects, including, for example, chronic malabsorption, risk of internal hernias, dumping syndrome, and weight rebound.
  • the combinatorial methods provided herein as exemplified in the Examples, provide results comparable to gastric bypass surgery, but without the need for invasive surgery.
  • This example describes and details exemplary combinatorial weight loss protocols and the results achieved.
  • the protocols are designed mimic the metabolic and physiological effects of bariatric weight loss surgery (z.e., gastric bypass surgery) by selecting combinations medications and treatments that mimic the metabolic and physiological effects of bariatric weight loss surgery (z.e., gastric bypass surgery).
  • a set of weight loss drugs are selected and combinations of the drugs are administered.
  • the combinations of drugs are a subset of the selected drugs. Different sets of combinations of the drugs can be rotated.
  • development of tolerance or desensitization to the medications/ drugs is avoided.
  • Weight loss comparable to or more than weight loss surgery, such as gastric bypass is achieved.
  • the combinatorial protocol exemplified below and described herein employs combinations of drugs include that peptides that are involved in regulation of the gutbrain axis. As shown herein, these protocols are more effective than the monotherapies.
  • a weight loss regimen described below, in which combinations of medications described below, were administered to human subjects.
  • the protocol was conducted in Punta Gorda, Florida. 96 subjects signed informed consent documents and were enrolled in the weight loss regimen. a. Subjects
  • the protocol was performed with the consent of each participant. Each participant understood that medications provided were being used “off label” and that they could opt out for standard of care, including monotherapy or counseling on diet and exercise. Benefits of the protocol were explained to the participants prior to initiation of the protocol, participants were counseled on general exercise, nutrition, and portion control, and medications were adjusted as needed. All subjects had failed standard of care protocols for weight management, which included intervention (z.e., surgical intervention) in some subjects.
  • Subjects were seen once weekly for the first 4 weeks, and then once every two weeks, for six visits, for a total of 10 visits over 16 weeks. Vital signs and side effects were assessed at each visit. No participants stopped the regimen during the initial 3- month period due to side effects. The most reported side effects include constipation (40% of participants), dry mouth (35% of participants), tremulousness (10%) and insomnia (5%). One male and one female were discontinued prior to completion of the first month due to social circumstances that prevented timely follow-ups. These subjects were excluded from the demographic information, set forth in Table 15, below.
  • Subjects were instructed to adhere to a liberal diet that included low carbohydrate and high protein in the ratio of 20% carbohydrates, 50% protein, and 30% fats, when possible. After 12 weeks, subjects were instructed to increase the proportion of carbohydrates. Subjects were not instructed to count calories; subjects were instructed to be cognizant of portion size/control. Estimated calorie count for the average subject ranged from approximately 1500-2000 kcal/day.
  • Weight and BMI are poor discriminators of health. Body weight, and, thus, weight loss can be composed of water, fat and muscle and at different ratios. The higher proportion of fat relative to muscle indicates an unhealthy state that can promote inflammation and insulin resistance. Conversely, having a higher ratio of muscle mass is associated with lower insulin resistance and improved insulin sensitivity (Srikanthan etal., J Clin Endocrinol Metab. (2011) 96(9):2898-903).
  • the combination therapy protocol in the Example assessed total weight loss, fat loss, and muscle loss/gain, which, in combination, is a more accurate predictor of health status (Gomez- Ambrosi, etal., Obesity 19, 1439-1444 (2011); Gomez -Ambrosi et al, Obesity 19, 1439-1444 (2011); Kim et al., Diabetes Care 34:504-506 (2011)).
  • Weight, fat and muscle mass were assessed using the InBody 520TM body composition analyzer (research grade).
  • the data presented below are based on analyses conducted with data from the InBody 520TM body composition analyzer (Biospace, Inc.; Los Angeles, CA USA), which assesses shifts of muscle to fat mass and shifts in water weight.
  • the InBody 520TM analyzer assesses weight, lean body mass, body fat mass, body water balance, body mass index (BMI), percentage of body fat, and muscle mass, using bioelectrical impedance. These parameters were assessed during the 36 weeks of the protocol. Subjects were weighed and assessed using InBody 520TM body composition analyzer at weekly intervals for the first four weeks, then once every two weeks for the completion of the initial 3 -month period. d. Combinatorial Pharmacological Regimen
  • Subjects were administered or self-administered the medications in the protocol by the appropriate route (z.e., subcutaneous injection or oral tablet/capsule) for 36 weeks.
  • Subjects initially were administered a combination of: 1) a GLP-1 agonist (sold under trademarks Trulicity®, B-cise®, Ozempic®, and Victoza®), 2) Phentermine, 3) Liothyronine, 4) topiramate (sold under the trademark Topamax® carbonic anhydrase inhibitor), and 5) Sermorelin.
  • the starting dosages are listed below in Table 17. The dosages were adjusted throughout the treatment period based on the subject response (z.e., weight loss), tolerability, volume of distribution, subject weight, and at the discretion of the treating physician.
  • the dosage of the GLP-1 agonists Trulicity® and Ozempic® was adjusted depending on the response to the medications.
  • the dosage of the Bydureon BCcise® GLP-1 agonist was not adjusted since it is only produced in a 2 mg injectable dose.
  • Phentermine was increased depending on subject response, to, for example, one 37.5 mg tablet in the morning and one-half tablet (18.75 mg total) at 2 p.m.
  • Liothyronine which had a starting dosage of 25 mcg, was increased to up to 100 mcg.
  • Topamax® carbonic anhydrase inhibitor which had a starting dosage of 25 mg, was increased to 100 mg as deemed appropriate.
  • SGLT2 is a low affinity sodium/glucose transporter
  • the regimen provided a minimum of four medications, and more medications were added and/or the dosages were modified as required in subsequent visits.
  • Table 17 Exemplary medications for regimens i. Exemplary Regimens
  • medications i-iv and ix listed above, were given initially and one or more of the initial medications was discontinued; Topamax® carbonic anhydrase inhibitor was the least tolerated, and was discontinued in some cases.
  • the regimen began with administration of medications i-iv and ix and other medications were added; additional medications were administered at week 2 or week 7, for example, if the subject was not responding and needed an extra pharmacological ‘push.’
  • the medication dosages were adjusted throughout the treatment period as needed, and as agreed to between the treating physician and the subject. For example, if a subject was losing at least four pounds per week, typically, no dosage change was made and no new medications were added. For larger subjects (z.e., starting weight over 300 pounds) who were losing less than 6 pounds per week, medication dosages were increased or additional medications were added to the regimen.
  • the hierarchy of decision making included 1) increase the dosage of the medications to the maximum allowable dosage or to the level of tolerability of the medication; and then 2) add a fifth or sixth medication, as needed to increase weight loss. ii. Representative Subjects
  • the regimens of a representative subset of subjects are set forth below.
  • the exemplary regimens also include examples of modifications made to the combination therapy, including the modifications described above.
  • Example For Example, subject 1, a male, received the medications set forth in Table 18, below (dosages are per day):
  • Table 18 Weight loss medications, phentermine, liothyronine, and a GLP-1 agonist
  • subject 2 a female, received the medications set forth in
  • Medications such as phentermine, and liothyronine were increased based on slower than desired initial weight loss.
  • phentermine was stopped and diethylpropion was rotated in, and subject took 25 mg diethylpropion per day for 4 weeks (weeks 13-16).
  • week 17 subject ceased taking Diethylpropion and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
  • Subject 2 previously had received a sleeve gastrectomy procedure for treatment of obesity. Following the procedure, Subject 2 regained weight and participated in the instant protocol to lose additional weight. Subject 2 had a starting weight of 168 pounds and weighed 142.6 pounds at the end of the 36-week weight loss protocol. Weight (pounds), fat and muscle loss of subject 2 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 21 below: Table 21:
  • subject 3 a male, received the medications set forth in
  • Weight loss medications (z.e., phentermine, liothyronine) were increased based on slower than desired initial weight loss. After 8 weeks on phentermine, phentermine was stopped and Mirabegron was rotated in, and subject took 25 mg Mirabegron every other day for 4 weeks (weeks 13-16). At week 17, the subject ceased taking Mirabegron and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
  • Subject 3 had a starting weight of 251.5 pounds and weighed 197.3 pounds at the end of the 36-week month weight loss protocol.
  • Weight (pounds), fat and muscle loss of subject 3 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 23 below:
  • subject 4 a male, received the medications set forth in
  • Weight loss medications ie., phentermine, liothyronine
  • Subject 4 had a starting weight of 489 pounds and weighed 395 pounds at the end of the 36-week month weight loss protocol. Weight, fat, and muscle loss of subject 1 after 12 weeks and 36 weeks on the weight loss protocol are set forth in
  • subject 5 a female, received the medications set forth in
  • Weight loss medications ie., phentermine, liothyronine
  • Phendimetrazine was rotated in, and subject took 25 mg Phendimetrazine every day for 4 weeks (weeks 13-16).
  • subject ceased taking Phendimetrazine and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
  • Subject 5 had previously received a gastric bypass procedure for treatment of obesity. Following weight loss from the procedure, Subject 5 regained weight and participated in the instant protocol to lose additional weight. Subject 5 had a starting weight of 188.5 pounds and weighed 149.3 pounds at the end of the 36-week weight loss protocol. Weight (pounds), fat and muscle loss of subject 1 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 27 below:
  • the results show that the average subject lost 31.5% fat at 36 weeks following the start of treatment.
  • Female subjects lost less total fat and percent fat than male subjects.
  • 36 weeks after the start of treatment female subjects lost an average of 12.1 kg, and 30.3% fat, and male subjects lost an average of 20.4 kg, and 31.5% fat.
  • Weight loss progressed over time in male and female subjects; total body fat loss continued to decrease from 21.4% and 22% at 12 weeks to 30.3% and 31.5% at 36 weeks for females and males, respectively.
  • the weight loss induced by the regimen described herein is similar to or greater than the 2-3 pounds (about 0.9-1.3kg) of weight loss per week or approximately 30 to 40 pounds (about 13-23 kg) the first 12 weeks after weight loss surgery.
  • the instant regimen shows results similar to a vertical sleeve gastrectomy (26.8 kg vs. 13-23 kg) in a population of subjects that are older and, thus, can have more challenges in losing body fat.
  • the instant regimen shows results that are better than mono- or dual therapy for treatment of obesity (see, e.g., Example above).
  • Total body weight lost includes fat weight, muscle weight and water weight.
  • the studies conducted herein also assessed the amount of fat and muscle loss, using the InBody 520TM body composition analyzer. Analysis of the bioelectrical impedance data shows that the percent body weight lost after treatment with the combinatorial therapy was not due to a concomitant loss in muscle mass. Subjects treated with the combinatorial therapy described herein lost 14% and 19% total body weight at 12 and 36 weeks, respectively. The muscle loss was only 2% and 3% at these time points, whereas the fat loss was 22% and 31.5% at these time points. These results indicate that at 12 and 36 weeks, the fat loss accounted for a greater percent of the total weight lost. Taken together, these results show that the combinatorial therapy for weight loss described herein, showed high overall weight loss and preferentially decreased fat compared to muscle.
  • This example compares the weight loss in subjects in the weight loss regimen described in the Example above using combinatorial pharmacological treatment described herein to other available pharmacological monotherapy treatments.
  • Wegovy® semaglutide weight loss medication for subcutaneous injection was approved by the U.S. Food and Drug Administration in June 2021, for chronic weight management in overweight and obese adults who also have at least one weight-related comorbidity (e.g., high blood pressure, type 2 diabetes, or high cholesterol).
  • Semaglutide is a GLP-1 incretin hormone that plays a role in appetite and digestion. Weight loss in subjects administered Wegovy® semaglutide weight loss medication was assessed previously in at least four studies (results detailed below in Table 30) studies 1, 3 and 4 were performed in non-diabetic subjects, and study 2 was performed in diabetic subjects. Study 1 was performed in subjects with obesity or overweight with a comorbidity.
  • Study 2 was performed in subjects with obesity or overweight with type 2 diabetes.
  • Study 3 was performed in subjects with obesity or overweight with a comorbidity who were undergoing intensive lifestyle therapy.
  • Subjects treated with Wegovy® semaglutide weight loss medication for 12 weeks lost an average of 5.7% of their body weight (combined results from studies 1-4).
  • Table 30 is adapted from HIGHLIGHTS OF PRESCRIBING INFORMATION for WEGOVY (semaglutide) injection, for subcutaneous use, Initial U.S. Approval: 2017, revised 06/2021, and sets forth the Wegovy® semaglutide weight loss medication studies 1-3:
  • LSMean least squares mean
  • CI confidence interval * pO.OOOl (unadjusted 2-sided) for superiority.

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Abstract

Delivery vehicles, such as liposomes, that display combinations for fat loss and muscle enhancing peptides for treating obesity and/or diabetes are provided. The delivery vehicles can be administered in combinatorial regimens, and administration in rotational combinatorial regimens.

Description

DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF Related applications
Benefit of priority is claimed to U.S. provisional application Serial No. 63/638,893, filed on April 25, 2024, entitled “DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF,” to inventors Jorge Luis Cabrera and Jorma A. Virtanen, and Applicant StarRock Pharma Inc.
Benefit of priority is claimed to U.S. provisional application Serial No. 63/675,214, filed on July 24, 2024, entitled “DELIVERY VEHICLES COMPRISING PROGLUCAGON DERIVED POLYPEPTIDES AND ANABOLIC POLYPEPTIDES AND USES THEREOF,” to inventors Jorge Luis Cabrera and Jorma A. Virtanen, and Applicant StarRock Pharma Inc.
This application is related to International PCT application No. PCT/US23/77508, filed on October 23, 2023, and published as International PCT publication No. WO2024/091863, published on May 02, 2024, entitled “COMBINATORIAL, AND ROTATIONAL COMBINATORIAL THERAPIES,” to inventor Jorge Luis Cabrera, and Applicant StarRock Pharma Inc.
Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING FILED ELECTRONICALLY
An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on April 19, 2025, is 64,041 bytes in size, and is titled 2702PCSEQ001.xml.
FIELD
Delivery vehicles comprising polypeptides for treating obesity are provided. Therapies include administration combinatorial regimens, and administration of rotational combinatorial regimens. The regimens comprise combinations of drugs and/or non-drug treatments. BACKGROUND
In the United States, there has been an unprecedented rise in overweight and obesity in the last decade. There are more obese US adults than those who are just overweight (Deng etal., Annual Review of Pathology (2016) 11(1): 421- 49). In 2008, the Journal of the American Medical Association (JAMA), reported that the obesity rate among adult Americans was estimated at 32.2% for men and 35.5% for women, which was confirmed by the CDC for 2009-2010. According to the CDC, “obesity is higher among middle-aged adults, 40-59 years old (39.5%) than among younger adults, age 20-39 (30.3%) or adults over 60 or above (35.4%) adults” (“Adult Obesity Facts,” published by the Centers for Disease Control and Prevention. Retrieved November 22, 2015). Projections indicate an increase in obesity prevalence to 60% in adult men, 40% in adult women, and 25% in children by 2050 (CDC Data Brief, 2014). The overweight and obesity epidemic undermines health, was formally classified as a “disease” in June 2013 by the American Medical Association, with much controversy.
Obesity is not confined to the United States. It is a global epidemic with serious medical and financial consequences. In 1997, the WHO formally recognized obesity as a global epidemic and viewed it as one of the most serious public health problems of the 21st century. Based on global trend data from 1975 to 2014, and an estimated world population of 8-9 billion, with the prevalence of obesity reaching 18% in men and 21% in women, almost a three-fold increase compared to prior to 1975. The Organization for Economic and Co-operation Development has projected an increase in obesity rates world-wide until at least 2030, especially in the United States, Mexico and England with rates reaching 47%, 39% and 35%, respectively (“Obesity Update 2017,” published by the Organisation for Economic Co-operation and Development. Retrieved 6 October 2018). Once considered a problem only of high-income countries, obesity rates are rising worldwide. Globally, there are now more people who are obese than who are underweight, a trend observed in every region over the world except parts of sub-Saharan Africa and Asia (World Health Organization, Obesity and overweight. Fact sheet updated June 2016. Geneva. Retrieved 22 Sept 2017).
There are treatments for obesity, but few that result in sustained weight loss and few that result in a loss of weight of more than 10% body weight. Hence, there is a need for treatments for obesity that result in sustained and continued weight loss to eliminate obesity.
SUMMARY
Provided are delivery vehicles for treating obesity and/or for treating type 2 diabetes. The delivery vehicles display combinations of polypeptides for weight loss, such as proglucagon polypeptides, and a polypeptide that prevents or reduces muscle loss associated with weight loss or promotes muscle growth (referred to as a muscle enhancing polypeptide). The delivery vehicles can contain or display other drugs, such as small molecule drugs, and they can contain the peptide drugs. Thus, the vehicles are for delivery of combinations of drugs, including small molecules and peptides. Also provided are combinations of delivery vehicles where the delivery vehicles each display a polypeptide and/or contain a small molecule drug, and a plurality of delivery vehicles are co-formulated or administered together so that the combination of at least two weight loss promoting peptides and a muscle enhancing and optionally a small molecule drug, such as an appetite inhibiting drug, are administered together. Devices for administering the delivery vehicles, such as syringes or pens, that contain single or multiple dosage amounts of the delivery vehicles also provided. The devices can contain multiple chambers, one containing a lyophilized composition containing the delivery vehicles or mixtures thereof, and a second containing a vehicle, such as PBS, for mixing with the delivery vehicles, for administration. A variety of the types of polypeptides linked to or embedded in the delivery vehicles can provided so that the combinations of polypeptides can be rotated, such as in regimens described in copending International PCT application No. PCT/US23/77508 (International PCT publication No. WO2024/091863). Provided are delivery vehicles for effecting treatment of obesity and/or diabetes, and for implementing the regimens and methods described in the copending application. The polypeptides described therein can be linked to or embedded in delivery vehicles, such as liposomes or extracellular vesicles or vehicles, such as exosomes, for administration. Two or three different polypeptides can be linked to or embedded in the delivery vehicle and administered, such as by injection, such as subcutaneous injection, and, depending upon the selected delivery vehicle by inhalation into the lungs or nose or by mucosal delivery, including orally. Most liposomal preparations and exosomal preparations cannot be formulated for oral administration, but there are some that can be so-administered.
The delivery vehicles or combinations for delivery vehicles contain or display a polypeptide for weight loss and a polypeptide for muscle enhancement (prevention or reduction of muscle loss associated with weight loss and/or increasing muscle mass). The delivery vehicles and composition include uses for treating obesity and/or diabetes, and/or other comorbidities, such as high cholesterol, heart disease, and hypertension, and other diseases, disorders, and conditions associated with metabolic syndrome.
All the methods and regimens and uses can provide the drug, generally a peptide drug, displayed on a delivery vehicle, such as a liposome or exosome. The peptides can be linked, by standard well-known methods, to the delivery vehicle or the delivery vehicle can be produced with the peptide incorporated into the surface. The delivery vehicles as detailed herein can display a plurality of peptides, or can display one or more and mixtures thereof can be administered or used together to provide a desired combination of peptides. In general, the methods, uses, and regimens herein are for treating obesity and/or diabetes. In general, the methods, regimens, and uses comprise a mixture of peptides for weight loss and for muscle enhancement for promoting or sustaining muscle growth, such as an anabolic peptide).
Provided are delivery vehicles, comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle; the surface of the delivery optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and/or have different activities; and the therapeutic peptides target pathways involved in obesity and/or diabetes, or have activity for treating obesity and/or diabetes. Also provided are compositions, comprising a mixture of delivery vehicles, where: each delivery displays at least one therapeutic peptide on the surface; and the composition comprises delivery vehicles selected so that the composition comprises at least three different displayed peptides. The composition can be formulated for any suitable route of administration, including, for example, intramuscular, intravenous, mucosal, parenteral, subcutaneous administration, oral, intranasal, inhalation, and other route. For example, the delivery vehicles and compositions are provided for subcutaneous administration. They can be provided as liquids or powders for reconstitution as a liquid.
Delivery vehicles include, but are not limited to liposomes, lipid nanoparticles, exosomes, and extracellular vesicles. The peptides displayed in or on the delivery vehicles comprise fat loss and muscle enhancement peptides; as noted muscle enhancement includes muscle loss reduction or prevention and/or increased muscle mass, such as an anabolic peptide. Exemplary of muscle enhancing peptides are sermorelin, tesamorelin, and IGF-1. The muscle enhancement polypeptide by enhancing muscle growth prevents or reduces the loss of muscle that is a problem with administration of GLP-1 agonists. For example, at least two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide. Polypeptides for fat loss can be selected from among: GLP-1, adiponectin, leptin, oxyntomodulin, PYY (peptide YY), amylin, pancreatic peptide, enterostatin/GIP (Gastroinhibitory Polypeptide), glicentin, glucagon, GRPP (glicentin-related pancreatic polypeptide), HGH (human growth hormone), CCK (cholecystokinin), neurotensin, secretin, IIP (myo-inositol 1 -phosphate), and MPGF (major proglucagon fragment). Peptides for muscle enhancement, include, but are not limited to, sermorelin, tesamorelin; and IGF1 (or HGH). For example, the combinations of drugs comprise peptides that comprise GLP-1, Oxyntomodulin, enterostatin/GIP (Gastroinhibitory Peptide); and Sermorelin. The liposomes and other delivery vehicles also can contain or display small molecule weight loss drugs, such as phentermine.
Delivery vehicles include liposomes, such as large multilamellar vesicles (LMV) and SMVs (small MVs). Liposomes include those that comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA). Phospholipids include synthetic and phospholipids from natural sources, such as egg yolks. Cholesterol can be added to the liposomes to improve properties, such as permeability. The molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less. Provided are delivery vehicles and compositions including liposomes comprising modified lipids. The liposome can include lipids modified with a reactive group for coupling with a peptide or with a peptide modified with a reactive group. In some liposome fabrication method and product embodiments, the reactive group for the coupling reaction is selected from among amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and/or alkyne reactive groups. In some embodiments, the reactive group is an amino group that forms an amide bond with an activated carboxylic ester, or is a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or is a hydrazide that bind with carbonyl groups, or is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprise azide and alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the peptide or protein the liposome.
Provided are delivery vehicles and compositions where the peptides are linked to the peptides via bonds formed by reaction of the reactive groups. For example, the delivery vehicle is a liposome and the peptide and/or liposome is/are PEGylated for linking the peptide to the liposome. The peptide and/or liposome can be PEGylated for linkage, or linked to or coated with streptavidin for reaction with biotin, such as biotin linked to the peptide. Provided are delivery vehicles and compositions, where the peptide and liposome are linked via an amide/peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2,3-triazole linker.
The linkage between the delivery vehicle, such as liposome, can comprise a spacer, such as, but are not limited to, a spacer that comprises polyethylene glycol (PEG) and/or and an oligonucleotides bound to the liposome and to the peptide linked to a complementary oligonucleotide. The delivery vehicle, such as a liposome, can comprise streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide. For example, the delivery vehicles, such as liposomes, are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form liposomes that display the peptides upon mixing these peptide derivatives with streptavidin liposomes. The linkage can include a spacer, such as a PEG spacer or oligonucleotide; one end of the spacer can be attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.
Provided are containers comprising the delivery vehicles or compositions provided herein. Containers include, for example, pens and syringes for administering the delivery vehicle.
Provided are pharmaceutical compositions comprising the delivery vehicles provided herein.
Methods of treatment of obesity and diabetes are provided. The methods comprise administering a delivery vehicle or composition provided herein. The methods can include the rotational combinatorial and rotational methods described herein, where the peptides are provided displayed on delivery vehicles, such as liposomes. The disease, disorder, or condition contemplated for treatment is/are obesity and/or diabetes. One combination of peptides can be administered, where the combination is displayed on the delivery vehicle. A plurality of combinations can be administered where the different combinations are provided on delivery vehicles. Mixtures of delivery vehicles displaying different peptides can be combined and administered. Corresponding regimens, as described herein are provided. Provided are methods and regiments and delivery vehicles that comprise combinations of peptides mimic effects of gastric bypass. For example, the methods and regimens include a combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin. The methods and regimens can further comprise a delivery vehicle that comprises a peptide that results in muscle enhancement, such as, for example, one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone. By virtue of muscle enhancement, the loss of muscle associated with weight loss treatment that accompanies weight loss is reduced or prevented. As a result, treated subjects, whether or not they exercise, have little or no muscle loss. The methods and regimens can comprise administration of a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP). Peptides in the methods and regimens and linked to delivery vehicles, such as liposomes, for example can be selected from among: GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosinetyrosine (PYY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and/or the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP -4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides. The delivery vehicles also can contain and/or display a small molecule weight loss drug, such as an amphetamine or appetite suppressant, such as phentermine. Exemplary of peptides that are displayed and small molecules that are incorporated into delivery vehicles are those selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alpha-glucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP-4) inhibitor), canagliflozin (sodium-glucose cotransporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin. For example, the peptides can be selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.
The methods and regimens and/or delivery vehicles further can comprise a mitochondrial uncoupler linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle. Exemplary of mitochondrial uncoupler include, for example, uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine). The small molecule drugs, which are available in oral dosage forms, such as phentermine, can be co-administered with the liposomes containing or displaying the polypeptides.
The methods and regimens herein include administering a plurality of different combinations of drugs, wherein: each combination of drugs is administered for a predetermined time; each combination of drugs is rotated until all combinations are administered at least once to comprise a cycle; a cycle comprises at least two different combinations; a combination comprises at least two different drugs that target different pathways or targets involved in the disease, disorder, or condition; each combination of drugs is unique among the combinations administered in a cycle, but a drug can be part of a plurality of combinations as long as the resulting combinations are unique; the disease, disorder, or condition is a chronic disease, disorder, or condition that requires treatment for at least 6 months; and the disease, disorder, or condition is not a cancer.
Delivery vehicles containing/displaying a combinations of the peptides, and optionally a small molecule(s), such as phentermine or a drug to treat a comorbidity of obesity, such as metabolic syndrome, diabetes, high cholesterol, heart disease, and hypertension, are provided. Examples of combinations of polypeptides for displaying on or delivering in a delivery vehicle, such as a liposome or an exosome include combinations of at least two or three weight loss drugs and a muscle enhancer, and can further include a small drug, such as an appetite suppressor, an amphetamine family drug, and/or drugs for treating obesity co-morbidities. The small molecules can be fabricated in association with the liposomes, or can be separately administered. Examples of fabrication of liposomes is detailed in the above examples, and known and/or apparent to those skill in the art. Exemplary combinations of peptides and sequences are as follow:
Drug 1 : GLPl/GIPl/Oxyntomodulin + ME* (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44 Drug 2: GLPl/GIPl/Amylin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44 Drug 3: GLP1/GIP1 /Glucagon + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44 Drug 4: GLP1/GIP1/CCK+ ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44 Drug 5: GLP1/GIP1/PYY + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 6 or 36 + SEQ ID NOs: 5, 8, 44 Drug 6: GLP1/GIP1 /Leptin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44 *ME= A muscle enhancer such as Sermorelin or Tesamorelin or IGF1 or other muscle enhancing peptide or drug that ameliorates the loss of muscle accompanying weight loss by promoting muscle growth
Different delivery vehicles can be rotated to provide rotational combinatorial treatment.
Provided are rotational combinatorial regimens for treating a disease, disorder, or condition, comprising a plurality of combinations of drugs and/or treatments for a disease, disorder, or condition, wherein: the disease, disorder, or condition has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the rotational combinatorial therapy comprises at least two different combinations of drugs and/or treatment; the drugs and/or treatments in each combination target different pathways or targets involved in the disease, disorder, or condition; each combination is administered at least once a cycle; each cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; the cycle for each combination can be the same or a different length of time; and at least one of the combinations comprises at least two different drugs that target different targets or pathways.
Provided are methods of treating a disease, disorder, or condition, comprising administering a drug regimen comprising serially administering two or more combinations of drugs and/or treatments, by administering the delivery vehicles provided herein, wherein: the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; the disease, disorder, or condition is a chronic condition that requires treatment for at least 6 months; the drug regimen comprises at least two combinations; at least one combination in the regimen includes at least two drugs that treat at least one target or pathway involved in the disease, disorder, or condition; the at least two drugs treat different targets, pathways, and/or have different activities.
Provided are combinatorial therapeutic methods of treating a disease, disorder, or condition, comprising administering a combination of at least 2 or 3 different drugs selected from among a plurality of drugs, and generally including a muscle enhancer; wherein the disease, disorder, or condition is a chronic disease, disorder, or condition; the disease, disorder, or condition is a disease, disorder, or condition that has more than one therapeutic intervention target or pathway for therapeutic intervention; each of the plurality of drugs can treat a target or pathway involved in the disease, disorder, or condition; each of the selected drugs has a different activity from the other drugs in the combination; and the combination reduces or eliminates desensitization to one or more of the selected drugs. Included are methods and regimens where desensitization results from downregulation of a receptor agonized by a drug in a combination, when it is administered as a monotherapy, or upregulation of a receptor antagonized by a drug in a combination, when it is administered as a monotherapy. In some embodiments, the methods and regimens are those where the drugs and/or treatments in each combination are administered simultaneously, sequentially, or intermittently. In administering the drugs/treatments in a combination they can be administered together, simultaneously, serially, or intermittently. They can be administered within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or longer, such as up to 2 or 3 days. Generally, the drugs and treatments in each combination are administered within 24 hours. The methods and regimens include those where the disease, disorder, or condition is selected from a disease, disorder, or condition that includes treatments that when used as a monotherapy, the subject becomes desensitized to the drug. The rotational methods herein can avoid desensitization. The methods and regimens can comprise at least 2, 3, or more cycles, whereby treatment lasts at least 6 months, 9 months, a year, or longer.
Among the embodiments provided herein are rotational combinatorial methods and regimens that comprise at least 3 different combinations in a cycle. Included are methods and regimens where each combination of drugs and/or treatments comprises at least 2 different drugs or treatments that target different pathways or targets for intervention. In some embodiments, the drugs and treatments in a combination or in more than one combination can target the same pathway or target for intervention.
In all embodiments, the cancer is not among the disease, disorder, or condition that is treated for which the regimen is employed.
Among the diseases, disorders, and conditions is obesity. Provided are methods and regimens for treating obesity. Provided are combinatorial methods and regimens for treating obesity, comprising administering a combination of at least three different drugs, wherein each targets a different pathway or different target for intervention for treatment of obesity. The methods and regimens can comprise rotating combinations of drugs, to thereby provide rotational combinatorial therapy. The methods and regimens include those where the combination(s) of drugs that is/are selected mimic the effects of gastric bypass. Provided are combinatorial weight loss regimens, comprising a combination of at least three different drugs, where the activity of the drugs mimics or has the activity of a peptide whose activity is altered following gastric bypass surgery. In general, in all embodiments or in at least one cycle of administration, a delivery vehicle comprising a muscle enhancing polypeptide, is administered to prevent or reduce loss of muscle associated with muscle or to enhance muscle growth.
Regimens for treating obesity comprising a combination or combinations of drugs whose effects or activities mimic the biological effects of gastric bypass surgery, such as methods and regimens where biological effects of gastric bypass comprise reduced absorption and/or malabsorption of food, decreased appetite, increased satiety, increased glycogenolysis and/or lipolysis, increased insulin sensitivity, modulation of energy expenditure, and inhibition of gastric emptying. Such methods and regimens can comprise or further comprise drugs that increase growth hormone, and/or promote or result in muscle enhancement so that at least the loss of muscle associated with weight loss is reduced or eliminated.
In accord with all of the methods and regimens provided herein, they can comprise combinations of drugs/treatments or selecting combinations of drugs and treatments, where the combinations of drugs and/or treatments are administered for a predetermined time of at least 1 week followed by administration of a different combination of drugs for a second predetermined time of at least a week, until all selected combinations of drugs are administered to complete a cycle; and repeating the same or a different cycle of combinations of drugs. For example, each combination can be administered for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least one month, or at least two months, or at least 3 months. Each combination can be administered for the same predetermined length of time, or each combination can be administered for a different length of time, or at least one of the combinations can be administered for a different length of time from the other combinations.
Provided are drug regimens for use for treating obesity, comprising a combination or combinations of drugs or drugs and treatments whose combined effects mimic gastric bypass, and optionally comprising additional drugs that promote or result in weight loss, wherein each combination comprises at least three different drugs that target a different pathway or intervention target involved in the etiology of obesity. The methods and regimens and uses can comprise one or more of a drug that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide- 1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and Glicentin; a drug that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; a drug that modulates energy expenditure comprising a drug selected from among leptin, oxyntomodulin, and glicentin; a drug that results in muscle enhancement comprising one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone; and a drug that promotes intestinal smooth muscle relaxation comprising vasoactive intestinal peptide (VIP).
The combinatorial methods, regimens and uses can be those where the drugs are selected from among drugs that have activities or effects selected from among: drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating or appetite, inhibit or modulate gastric acid secretion, limit or decrease the rate of gastric emptying, enhance muscles, increase glycogenolysis, increase insulin sensitivity, enhance the body weightlowering and/or glucose-lowering efficacy of GLP-1, medication(s) or therapy that decrease ghrelin or ghrelin-associated activation pathways, and drugs and treatments that reduce or antagonize ghrelin. Exemplary combinations of drugs include combinations of drugs that are selected from among GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin/glucose-dependent insulinotropic peptide or gastric inhibitory polypeptide (GIP,) cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (ECA), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15). Other exemplary combinatorial methods, regimens, uses include those where the drugs that are combined comprise a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP-4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, drugs that increase gastric inhibitory polypeptide (GIP2), and mitochondrial uncouplers, such as, but not limited to those in which the drugs are selected from among one or more of dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin; those where drugs in a combination or the combinations are selected from among a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant. The methods, regimens, and uses include those comprising a mitochondrial uncoupler, such as, for example, where the mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM 15 ((2 -fluorophenyl) { 6- [(2-fluorophenyl)amino]( 1 ,2,5 -oxadi azolo [3 ,4e] pyrazin-5-yl)} amine). The small molecule drugs, which are available in oral dosage forms, such as phentermine, can be co-administered with the liposomes containing or displaying the polypeptides.
For all of the methods, regimens, and uses the combination or each combination of drugs comprises at least three drugs that target different pathways or targets for intervention. These include those in which the disease, disorder, or condition requires treatment for at least 6 months, 9 months, or 1 year, or longer, or longer and indefinitely, or for life.
Provided are methods for preventing (or reducing the risk) of desensitization to treatments for a disease, disorder, or condition, comprising administering a rotational combinatorial therapeutic regimen. Such methods employ the methods and regimens and uses described above and elsewhere herein. As described the combinations of drugs and treatments can be administered together, sequentially, intermittently, and/or within a predetermined time period, such as within up to a 24- hour period, or up to a 12-hour period, or up to a 6-hour period or less.
Exemplary of the methods, regimens and uses is a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance muscles, and inhibit gastric emptying; b) a second combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and promote muscle enhancement.
Provided are rotational combinatorial regimens and methods and uses for treatment of obesity comprising at least three different combinations per cycle, wherein: a) a first combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance muscles, and inhibit gastric emptying; b) a second combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote muscle enhancement, and increase glycogenolysis; and c) a third combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, enhance the body weight-lowering and glucose- lowering efficacy of GLP-1, and promote muscle enhancement. For example, any of the methods, regimens, and uses herein can be a rotational combinatorial regimen for treating obesity, comprising administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, and enhance or promote muscle. For example, a method, use, or rotational combinatorial regimen for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and/or lipolysis, reduce eating, reduce gastric acid secretion, limit the rate of gastric emptying, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination that comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination that comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-and lowering efficacy of GLP- 1, and enhance or promote muscles. In another embodiment that is a rotational combinatorial regimen for treating obesity, the regimen, method, or use comprises the following combinations or administering a rotational combinatorial regimen comprising the following combinations: a) a first combination comprising drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprising drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprising drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement. In other embodiments, the rotational combinatorial regimen, method, or use for treatment of obesity comprises at least three different combinations per cycle, wherein: a) a first combination comprises drugs that increase insulin sensitivity, accelerate glycogenolysis and lipolysis, enhance the body weight-lowering and glucose-lowering efficacy of GLP-1, promote or result in muscle enhancement, and inhibit gastric emptying; b) a second combination comprises drugs that suppress appetite, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, decrease food intake and body weight, promote or result in muscle enhancement, and increase glycogenolysis; and c) a third combination comprises drugs that inhibit gastric motility, increase insulin sensitivity, accelerate glycogenolysis and lipolysis, reduce eating and gastric acid secretion and limit the rate of gastric emptying, and promote or result in muscle enhancement.
In all embodiments herein, the method or regimen or use can comprise combinations in each combination or in each cycle administered for 2-, 3-, 4-, 5-, or 6-month intervals. The intervals between or for each administration of each combination are the same or vary. In accord with the methods, regimens, and uses, at least one combination in a cycle can comprise at least three different drugs. For example, at least one combination in a cycle comprises at least four different drugs; and optionally at least one combination includes only a single drug.
In particular, provided are delivery vehicles, including liposomes and exosomes, compositions, regimens, methods, uses, combinations, kits, and articles of manufacture as follows and/or including variations thereof within the skill in the art. Provided are delivery vehicles, comprising a combination of therapeutic peptides, where: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of delivery vehicle or in the delivery vehicle; the surface of the delivery vehicle optionally is modified for linkage of the polypeptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and/or have different activities; and the peptides target pathways involved in obesity and/or diabetes, or have activity for treating obesity and/or diabetes or other obesity comorbidity. These vehicles can further comprise a small molecule drug for treatment of obesity or an associated comorbidity.
Provided are compositions, comprising a mixture of delivery vehicles, where: each delivery vehicle displays at least one of the weight loss or fat loss peptides on the surface and/or contains the at least one peptide; and the composition comprises delivery vehicles that are selected so that the composition comprises at least three different peptides. The compositions can be formulated for a suitable route of administration, including injection, oral, and intravenous. For, example, the delivery vehicles are administered by subcutaneous (SC) injection in a volume of less than about 5 mL or 3 mL, or in a volume of 10 mL or more, particularly if formulated with an excipient the facilitates administration, such as a hyaluronidase.
Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles (LPNs), exosomes, and other extracellular vesicles.
The peptides in or on the delivery vehicles or in compositions or for combination therapy or for administration with the delivery vehicles comprise fat loss and muscle enhancement peptides, wherein muscle enhancement polypeptides reduce or eliminate loss of muscle mass or increase muscle mass. Additional drugs for administration include small molecule drugs, which can be incorporated in or on or associated with the delivery vehicle (such as by non-covalent interaction), for weight loss, for treatment of comorbidities, such as hypertension, high cholesterol, and diabetes, associated with obesity, and or for treating other diseases, disorders, and conditions.
Exemplary of the delivery vehicles are those where two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide. For example, the polypeptides for fat loss (PeptideFL) are selected from among: PeptideFL 1=GLP-1,
PeptideFL 2=Adiponectin,
PeptideFL3=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide), PeptideFL9= Glicentin, PeptideFL 10 = Glucagon, PeptideFL 11=GRPP, PeptideFL 12=HGH 176-191, Peptide FL13= CCK, PeptideFL 14= Neurotensin, PeptideFL15= Secretin, PeptideFL 16= IP1, and
PeptideFL17= MPGF (major proglucagon fragment); and optionally a muscle enhancing polypeptide and/or a drug such as a myostatin pathway inhibitor, such as an antibody, such as bimagrumab, that inhibits muscle wasting. In embodiments, the peptides for muscle enhancement (PeptideME) are selected from among: PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and PeptideME2=IGFlor human growth hormone to induce IGF1. Alternatively, in place of or in addition to the ME drugs, a myostatin pathway inhibitor can be included, such as for example, an antibody or an antigenbinding fragment thereof that competes with apitegromab for antigen binding. Other myostatin pathway inhibitors include, but are not limited to, a ligand trap (e.g., ACE- 031, ACE-083, and BIIB-110/ALG-801); an anti-ActRIIb antibody (e.g., bimagrumab); a neutralizing anti-myostatin antibody (e.g., stamulumab (MYO-029), domagrozumab (PF-06252616), or Landogrozumab (LY2495655)), a myostatin peptibody (e.g., AMG-745/PINTA-745), or an anti -myostatin adnectin (e.g., RG6206 or BMS-986089 (also known as taldefgrobep alfa)); wherein, further optionally, the non-selective myostatin pathway inhibitor also inhibits Activin A and/or GDF11 (see, US20240368262, which describes myostatin pathway inhibitors).
In other embodiments, the peptides for delivery and/or administration are peptides for fat loss (FL) and optionally peptides for ME, where: a) the polypeptides for fat loss are selected from among:
PeptideFL 1=GLP-1,
PeptideFL 2=Adiponectin,
PeptideFL3=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide),
PeptideFL9= Glicentin,
PeptideFL 10 = Glucagon,
PeptideFL 11=GRPP,
PeptideFL 12=HGH 176-191,
Peptide FL13= CCK,
PeptideFL 14= Neurotensin,
PeptideFL15= Secretin,
PeptideFL 16= IP1, and
PeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement are selected from among:
PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and
PeptideME2=IGFl (or human growth hormone to induce IGF1).
Exemplary combinations include those, for example, where the peptides comprise GLP-1, Oxyntomodulin, enterostatin/GIP (Gastroinhibitory Peptide); and Sermorelin, and other combinations as detailed herein.
In all embodiment, including liposomes, exosomes, and LPN, and extracellular vesicles, the peptides and other drugs can be exogenously introduced into and/or onto the vehicle or fabricated with the vehicle so that the peptide/drug is associated with or in the vehicle by any suitable interaction, including covalent bonding and non-covalent interactions.
Delivery vehicles, include, but are not limited to, liposomes, including liposomes that are large multilamellar vesicles (LMVs). For example, the liposome can comprise phospholipids, such as, for example, one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA). Liposomes can be from the phospholipid is from a natural source and loaded with the peptides and drugs or reacted to become associated with the peptides and other drugs, such as by covalent and/or non-covalent interactions. The liposomes can cholesterol, particularly so that the amount of cholesterol is sufficient to increase the permeability of the liposome compared to the liposome that does not contain the cholesterol, such as for example, where the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less. The liposome or other vehicle can lipids or other molecules modified with a reactive group for coupling with a peptide or peptide modified with a reactive group, such as for coupling with a peptide or peptide modified with a reactive group. Exemplar reactive groups for the coupling reaction include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne groups. The liposome can comprise lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group. Reactive groups include, but are not limited to, an amino group that forms an amide bond with an activated carboxylic ester, or a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or a hydrazide that binds with carbonyl groups, or a is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprises azide and/or alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the protein to the liposome. Other delivery vehicles and peptides can be similarly modified as appropriate. In the resulting delivery vehicles, the peptides can be linked to the vehicle via the bonds formed by reaction of the reactive groups. For example, provided are liposomes or other vehicle, where the peptide and liposome (or other vehicle) are linked via an amide/peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2, 3 -triazole linker. In other embodiments the peptide and/or vehicle, such as the liposome, is/are PEGylated for linking the peptide to the liposome. In some embodiments that the linkage of the peptide to the delivery vehicle, such as the liposome, comprises a spacer, such as, but are not limited to, polyethylene glycol (PEG) and/or and an oligonucleotides bound to the vehicle, such as a liposome, and to the peptide linked to a complementary oligonucleotide. In other embodiments that vehicle, such as the liposome, comprises streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide. In other embodiments, the vehicle, such as the liposomes, are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form vehicles, such as liposomes, that display the peptides upon mixing these peptide derivatives with streptavidin liposomes. The linkage can comprise a PEG spacer, where one end of the spacer is attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.
Also provided are combination of the delivery vehicle or compositions provided herein that comprises one or more small molecule drug(s), where: the small molecule drug enhances weight loss or treats a comorbidity associated with obesity; and the small molecule is formulated in or with the delivery vehicle or is for administrations separately from the delivery vehicle. Exemplary small molecule drug(s) is/are selected from among one or more of: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate. The delivery vehicle and small molecule drugs can be for administration together in a single composition or for administration in separate compositions for administration at substantially the same time, sequentially, or intermittently, or in accord with a particular combination therapy regimen, in which the drugs are administered in a particular order and/or according to a particular schedule. Regimens for administration of the combinations of the drugs/peptides are provided.
Also are provided are containers that comprise the delivery vehicle or compositions or combinations provided herein. Exemplary of containers are vials, pens, and syringes, including injectors, such as autoinjectors. Autoinjectors are known that provide for self-administration. Included are autoinjectors are multicompartment containers, where one compartment contains the delivery vehicles or a mixture of different delivery vehicles. They can be provided in a pharmaceutically acceptable vehicle or can be lyophilized. In some embodiments the peptides/drugs are provided as lyophilized powders, and there is a separate compartment that contains the pharmaceutically accept vehicle for solubilizing or dissolving the powders to produce a solution or suspension, such as microemulsion, suitable for injection, particularly subcutaneous injection.
A pharmaceutical compositions containing any of the delivery vehicles and mixtures there in a pharmaceutically acceptable vehicle for administration are provided. These compositions for administration or for use in methods for treating obesity and/or associated diseases, disorders, and conditions. Hence, methods for treating obesity as well as other comorbidities and uses for the vehicles, compositions, and combinations are provided. For example, provided are methods and uses for treatment of obesity or diabetes in which a delivery vehicle, and/or composition and/or combination provided herein is/are administered. This includes combinatorial and rotational methods, such as those, where the drugs include peptides and optionally small molecule drugs; and the disease, disorder, or condition is obesity and/or diabetes; where at least two combinations are rotated for each cycle of treatment; and treatment comprises at least two cycles.
Regiments for treating a disease, disorder, or condition, comprising combinations of peptides for use in a rotational combinatorial regimen are provided, such as where the disease, disorder, or condition is obesity or diabetes, or other obesity comorbidity, or other chronic disease, disorder, or condition that requires treatment for at least 6 months; each combination comprises at least two different peptides that target different pathways or intervention targets; the combinations of peptides comprise a delivery vehicle or composition provided herein. The regimen can include a small molecule drug or drugs for weight loss and/or for treating a comorbidity associated with obesity or other chronic condition. For example, the small molecule drug can be selected from one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin. In accord with the regimen, the small molecule drug can be incorporated in or on the delivery vehicle and/or the small molecule drug is for co-administration, simultaneously, sequentially, or intermittently with the delivery vehicle(s). The methods can be a rotational combinatorial method in which combinations of peptides and/or drugs are rotated. Such methods and regimens can comprise a plurality of cycles of administration of different combinations of peptides and optionally the small molecules, where each combination is administered at least once a cycle; a cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; and at least one of the combinations comprises at least two different drugs that target different targets for intervention or pathways. For example, each cycle can comprise one delivery vehicle that comprises at least 3 different peptides and optionally small molecule drugs, or mixtures of delivery vehicles that comprise different peptides and/or small molecule drugs, each delivery vehicle comprises at least peptide, whereby at least three different peptides are administered in each cycle; and the different delivery vehicles are administered together or serially. The small molecule drugs include, for example, one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin. Different vehicles or combinations of vehicles and/or drugs can be administered within 24 hours or other suitable time period determined by the skilled person, of each other.
In the delivery vehicles, methods, and/or regimens, each peptide can be selected to targets a different pathway or different target for intervention for treatment of obesity. For example, the combinations of peptides mimic all or some of the effects of gastric bypass. Exemplary combinations of peptides include, but are not limited to, combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide-1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin.
In accord with the descriptions of the delivery vehicles, methods and regimens provided herein, the vehicle can comprise and/or display and/or contain combinations of peptides selected from among combinations of drugs for fat loss + a drug for muscle enhancement as follows: a) GLP1 /GIP 1 /Oxyntomodulin + Sermorelin or Tesamorelin or IGF1; b) GLPl/GIPl/Amylin + Sermorelin or Tesamorelin or IGF1; c) GLP1 /GIP 1 /Glucagon + Sermorelin or Tesamorelin or IGF1; d) GLP1/GIP1/CCK+ Sermorelin or Tesamorelin or IGF1; e) GLP1/GIP1/PYY + Sermorelin or Tesamorelin or IGF1; and f) GLP1 /GIP 1 /Leptin + Sermorelin or Tesamorelin or IGF1. Exemplary amino acid sequences of each of the peptides for each of a)-f) are as set forth set forth in the following SEQ IDs, or are portions, or are variants thereof that have at least 90% or at least 95% sequence identity thereto and retain activity for effecting fat loss or muscle enhancement: a) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44; b) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44; c) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44; d) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44; e) SEQ ID NOs: 1, 10, 6 or 36 + SEQ ID NOs: 5, 8, 44; and f) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44.
In embodiments herein the delivery vehicle, composition, combination, method, and/or regimen includes a peptide that results in muscle enhancement or can include a peptide or other molecule that inhibits or reduces muscle loss, such as a myostatin pathway inhibitor. Exemplary muscle enhancement peptides and drugs include, but are not limited to, one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone. The herein the delivery vehicle, composition, combination, method, and/or regimen can comprise a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).
Peptides can be selected from among: a) GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (EC A), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and/or b) the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase-R (DPP -4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides.
Exemplary of embodiments herein are those in which the peptides and drugs are selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alphaglucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin (SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin. In other embodiments of the delivery vehicles, methods, regimens, compositions, and combinations, are those where the peptides and drugs are selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP -4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant. In all embodiments herein, the delivery vehicle, composition, method, use, or regimen can include or further comprise a mitochondrial uncoupler, wherein the mitochondrial uncoupler is provided linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle, such as a mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2- Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine).
It is understood that those of skill in the art can fabricate or obtain the peptides, and delivery vehicles, and/or fabricate the delivery vehicles by known methods. Nevertheless, methods for preparing or fabricating the delivery vehicles and/or components thereof are provided. Included are methods for preparing a PEGylated peptide, comprising preparing a PEGylated lysine and adding it to peptide during solid phase synthesis, where optionally the PEGylated lysine is prepared by reacting the epsilon-amino group of the lysine with a carboxyl terminated PEG moiety via an amide group to produce a PEGylated lysine. The peptides can be modified by replacing lysines that are not intended to be PEGylated with a conservative amino acid. The methods of synthesis can include adding the PEGylated lysine to a peptide during solid phase synthesis of the peptide. The PEG moiety comprises a functional group for conjugation with a reactive group, such as one on a liposome. Functional groups include, but are not limited to, acetylene that is reacted with azide in the liposome (click chemistry). Other methods of conjugating a polypeptide to a liposome or other delivery vehicle, comprise: adding a lysine to a polypeptide during solid phase synthesis by reacting the epsilon-amino group of lysine with a carboxyl terminated PEG moiety via an amide group to produce a peptide comprising a lysine comprising the PEG moiety, which comprises a functional group for conjugation with the liposome; and conjugating the PEGylated peptide to the liposome. The functional group, such as acetylene, can be reacted, for example, with azide fabricated in or added to the delivery vehicle, such a liposome, thereby employing click chemistry. Any of the delivery vehicles provided herein can be so-prepared, including where the peptide is a GLP-1 pathway agonist and/or a muscle enhancer peptide, or any other combinations as described herein or that are apparent to the skilled artisan from the description herein. The method can include prepared at least three pegylated peptides and linking the peptides to one delivery vehicle, such as a liposome, or each to a different delivery vehicle, such as a liposome, or two can be lined to one delivery vehicle, such as a liposome, and the third on a separate delivery vehicle, such as a liposome, or other combinations, or all can be linked or introduced into or onto a single delivery vehicle, such as a liposome. Exemplary thereof are wherein the peptides linked to the delivery vehicles, such as a liposome, or to different delivery vehicles, such as liposomes, wherein a least two of the peptides are for fat loss, and one is for muscle enhancement. For example, peptides can be selected where: a) the peptides for fat loss (FL) are selected from among:
PeptideFL 1=GLP-1,
PeptideFL 2=Adiponectin,
PeptideFL=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide),
PeptideFL9= Glicentin,
PeptideFL 10 = Glucagon,
PeptideFL 11=GRPP,
PeptideFL 12=HGH 176-191,
Peptide FL13= CCK,
PeptideFL 14= Neurotensin,
PeptideFL15= Secretin,
PeptideFL 16= IP1, and
PeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement (ME) are selected from among: PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and
PeptideME2=IGFl (or human growth hormone to induce IGF1).
Provided are delivery vehicles, such as liposomes that comprise one or more of a fat loss peptide and a muscle enhancement peptide produced by any of the above methods or methods that in light of the disclosure herein are known to the skilled artisan.
In all embodiments herein, the delivery vehicle, composition, use, method, regimen, or liposome, can include a small molecule drug, such as where the drug is for weight loss and/or a co-morbidity associated with obesity. Comorbidities include, but are not limited to, diabetes, hypertension, high cholesterol and/or other elevated lipids (dyslipidemia), metabolic syndrome, and heart disease. Small molecule drugs include, but are not limited to, Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
In all embodiments herein, the products and methods can include phentermine, including regiments and methods where the phentermine is provided in or on the delivery vehicle, or wherein phentermine is provided as a separate composition, such as in a tablet, capsule, gel, or other form for oral administration, for combination therapy. In all embodiments, or the delivery vehicles, compositions, combinations, uses, method, regimen, the delivery vehicle can be an extracellular vesicle or an LPN, or an exosome, or a liposome.
Also provided are kits and articles of manufacture containing the delivery vehicles and containers and compositions and combinations.
Brief Description of Drawings
Figure 1 depicts bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1/3 and PC2.
Figures 2A and 2B depict a liposome with linkages and linked moieties. Figure 3 depicts PEG (X) with an N-hydroxy succinimide active ester on one end. Figure 4 depicts an exemplary coupling chemistry in which X is Br or I.
Figures 5A-C depict a polypeptide with free lysines and Pegylation thereof. Figure 5A depicts a short segment of a polypeptide, such as any of the GLP-1 agonist polypeptides and/or a muscle enhancement polypeptide, which is Pegylated as shown herein, and Figure 5B depicts an exemplary resulting product in which a lysine is PEGylated. As an example, the PEG linked to the lysine has an azide group at the end for coupling to a liposome that has an acetylene group on the surface. Figure 5C depicts the product.
OUTLINE
A. DEFINITIONS
B. OVERVIEW
C. Delivery vehicle -components
1. Component -Proglucagon derived Peptides (PGDP) and muscle enhancement peptides a. Proglucagon Derived Peptides and tissue specific secretion b. Unimolecular multi-agonists c. Enhancing fat loss efficacy via proglucagon derived peptides
(PGDP) and co-secretory molecules released after a RYGBP
2. Delivery vehicles
3. Conjugation/Binding of peptides on the liposomes
4. Spacers
5. Linkage of peptides to the Delivery vehicles a. PEGylation of Peptides for linkage to lipid particles, such as liposomes b. Synthesis of Pegylated peptides for conjugation to liposomes c. Liposome preparation for linking to the peptides
D. FORMULATIONS AND ROUTES AND MODES OF DELIVERY
E. COMBINATORIAL THERAPY AND ROTATIONAL COMBI NA-'TORIAL THERAPY
1. Identification of diseases, disorders, or conditions for treatment a. Disease, Disorder, or Condition with a Plurality of Known Treatments b. Chronic Conditions c. Conditions where Patients Develop a Tolerance to Treatments
2. Development of a Combinatorial Rotational Therapy Regimen
1) Identify known treatments/therapies for each disease state or pathways associated with each disease state
2) Identify the pathways, mechanism of actions or targets
3) Select treatments/therapies that activate different pathways, have different mechanism of actions or targets, and/or that are compatible with a rotational therapy
4) Identify combinations that include at least 2 therapeutics known to activate different molecular and/or cellular pathways and design a regimen for administration of the combinations 5) Create a regimen to administer multiple rounds of treatment, with combinations of therapeutics
F. METHODS OF TREATMENT AND USES
1. Therapeutic Uses of the Combinatorial Therapy a. Combination Therapies in Cancer b. Combination Therapies in Pain Management c. Combination Therapies in Oral Contraception d. Combination Therapies to Treat Pathogens e. Combination Therapies to Treat Alzheimer’s Disease f. Combination Therapies to Treat Hypertension g. Combination Therapies to Treat Parkinson’s Disease h. Combination Therapies to Treat Chronic Obstructive Pulmonary Disease (COPD) i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions j. Combination Therapies to Treat Overweight and Obesity
G. COMBINATORIAL AND ROTATIONAE COMBINATORIAL THERAPY FOR WEIGHT EOSS
1. Limitations of Existing Treatments for Weight Loss
2. Obesity and the Challenges of Treatment
3. Pharmacological Treatments a. Amphetamines (e.g., phentermine-topiramate) b. Lipase inhibitors (e.g., Orlistat) c. Serotonergic agonists - Neuromodulators (e.g., lorcaserin) d. Bupropion/Naltrexone (such as the product sold under the trademark Contrave®) e. Glucagon-like peptide-1 receptor (GLP1R) agonists f. Mitochondrial uncouplers g. Thyroid hormones h. Drug cocktails i. Cannabinoid receptor antagonists j. GIPR agonists and GIPR/GLP1R combination agonists k. GLPIR/glucagon dual agonists l. Summary
4. Surgical Treatments
H. MODIFICATIONS AND ENHANCEMENTS OF PHARMACOLOGICAL WEIGHT LOSS TREATMENTS TO IMPROVE CLINICAL OUTCOMES
1. Developing Combination and Rotational Combinatorial Therapies for Weight Loss
2. Pathways to Target for Weight Loss and Exemplary Polypeptides a. Therapeutic Combinations and Regimens b. Combination Drug Therapy to Mimic Gastric Bypass I. PHARMACEUTICAL PRODUCTION, COMPOSITIONS, AND FORMULATIONS
1. Formulation and Administration of the Combinatorial Therapy
2. Dosage Forms
3. Dosage and Administration
4. Dosage and Administration for Treating Obesity and Overweight
5. Routes of Administration of the Combinations a. Administration of Combinatorial Treatments for Improved Weight Loss
6. Articles of Manufacture and Kits
J. METHODS OF ASSESSING ACTIVITY, BIOAVAILABILITY AND PHARMACOKINETICS
K. SEQUENCE SUMMARY
L. EXAMPLES
A. DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong(s). All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. If there is/are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, a delivery vehicle refers to macro-molecular structures in an emulsion, such as micelles, inverse micelles, lipid bilayers (liposomes) and cubosomes or a mixture thereof, as well as lipid nanoparticles and other lipid-based vehicles. Delivery vehicles include, but are not limited to, liposomes, lipid nanoparticles, extracellular vehicles, such as exosomes. The polypeptides are displayed on the surface of the vehicles such as by linkage to moieties on the surface. In other embodiments, the polypeptides can be embedded in the surface when the delivery vehicle is formed; and/or can be expresses in a membrane protein in vivo, such as during formation or preparation of an extracellular vehicle. As used herein, therapies used interchangeably with treatments include drugs and can include other non-drug treatments. Thus, treatments include drugs (or medications) and non-drug treatments, such as, for example, surgery.
As used herein, drugs include any administered therapeutic and is used interchangeably with medications.
As used herein, a therapeutic agent is used interchangeably with therapeutic and includes, but is not limited to: conventional drugs and drug therapies; vaccines; pharmaceutical medications; pharmaceuticals; homeopathic medications; peptides; protein therapeutics; radio-protectants; radiation therapy; and chemotherapeutics. A therapeutic agent can effect amelioration of symptoms of a disease, disorder, and/or condition or can prevent or reduce the risk of developing a disease, disorder, or condition, or reduce the severity of disease, disorder, or condition. The combinations when used for the combinatorial therapy as described herein include at least 3 treatments, such as three different therapeutics. For combinatorial rotational therapy combinations that are rotated, the combinations can include 2 treatments in a combination or combinations of treatments. Some of the rotations can include a single drug or therapy, as long as one or more others rotational combinations include at least two. Generally, each rotated combination includes at least two different drugs or treatments.
As used herein, combinatorial therapy or combinatorial protocol or regimen refers to combinations of treatments for a disease, disorder, or condition from among a plurality of treatments, at least 2 and generally at least 3, where each treatment acts on or interacts with or modulates a different target molecule and/or target pathway involved in or that mediates the disease, disorder, or condition.
As used herein, a rotational combinatorial therapy is used interchangeably with combinatorial rotational treatment, or rotational combinatorial therapy, or rotational combination therapy, or rotational combination treatment or CRT, or grammatical variations thereof. A rotational combinatorial therapy or protocol or regimen includes at least two different therapeutic combinations at least one of which, and generally each of which, includes at least two therapeutics or therapies known to activate different molecular and/or cellular pathways or targets involved in a disease, disorder, or condition. Combinatorial rotational therapy, thus, is a combinatorial therapy protocol that includes a plurality of rounds of treatment with different combinations of treatments or with the same combination but not successively. The different combinations are therapeutics are administered for a predetermined time, and then rotated for another predetermined time. The number of rotations of combinations can be two, three, four, five or more, and can be administered for weeks, months, years, and indefinitely depending upon the disease, disorder, or condition. The predetermined time can be days or weeks or months, generally is about 2-6 weeks. Each predetermined time is not necessarily the same.
As used herein, a rotational combinatorial pharmacological treatment (also referred to a rotational combinatorial therapy refers to a combinatorial therapy or treatment with pharmaceuticals or medications or drugs or treatments in the combinations where combination are rotated. The rotational combinatorial pharmacological treatment includes a plurality of combinations of a plurality of pharmaceuticals or medications or drugs that are rotated in accord with a rotational regimen or protocol. The rotational regimen generally is employed to prevent desensitization to a particular drug. A rotational combinatorial regimen herein includes two or more different therapies or therapeutics (e.g., combinations of therapies and/or therapeutics) that are administered such that the combinations of the therapies are rotated for a plurality of rounds of treatment. Where there is a plurality of rounds of treatment, at least one round can include only a single treatment. Generally, for rotational combinatorial protocols, the rounds include combinations of at least two different treatments, where each targets a different pathway involved in the disease, disorder, or condition.
As used herein, rotate or rotational or grammatical variations thereof include an exchange of one treatment for another. A combinatorial rotation refers to changing one therapeutic combination to another, which has the effect of improving the therapeutic response, such as by avoiding desensitization to a therapeutic of combination thereof. This also can reduce adverse side effects, and/or the severity of adverse side effects. In examples herein, a rotation includes a regimen in which a first combination of more than one therapeutics is administered, and the therapy is rotated (or switched) to a second combination one or more one therapeutic, and, optionally, the therapy is rotated (or switched) to a third combination of more than one therapeutics. Generally, the rotations occur according to a predetermined time schedule, or in view of a physician’s judgement, such as based on an observation that a combination has reduced effect. In some examples, the therapies are rotated more than three times, such as more than four times, more than five times, more than six times, more than seven times, more than eight times, more than nine times, or more. In some instances, such as chronic diseases, disorders, and conditions, the rotation of therapies continues for up to life.
As used herein, a regimen is used interchangeably with protocol and refers to a course of medical treatment. In some examples the regimen is designed to improve or preserve the health of the patient or to attain a particular result. A therapeutic regimen or protocol includes the timing for administration of the therapeutics, timing for cessation of therapeutics, therapeutic dosage(s), the particular therapeutics that are included in particular combinations, and other components of experimental design, such as how therapeutics are administered. A protocol can include provisions for how the protocol can be modified, such as, for example, in the case of receptor downregulation or an increase in adverse side effects.
As used herein, a cycle is generally a series of events that are repeated regularly in the same order. A cycle, with reference to a rotational combinatorial regimen, refers to the repeated administration of combinations of drugs, where a combination of drugs (or a drug) is administered for a period of time followed by another combination of drugs, and so on until all combinations in a cycle of treatment are administered. The cycle then can be repeated; generally, the same combinations of drugs or treatments are administered. It, however, is within the discretion of the physician to change the drugs/treatments in accord response(s) of the treated subject, such as to improve therapeutic effectiveness, eliminate or reduce desensitization, and/or for other reasons, such as to reduce adverse side effects. In accord with a rotational regimen, a combination can include a single drug, as long as at least one combination in the cycle includes two or more drugs or treatments.
As used herein, disease or disorder or condition refers to a pathological or undesirable or undesired state in an organism resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and that is characterized by identifiable symptoms. Obesity and overweight are conditions characterized by excess body weight.
As used herein, mucosal delivery refers to delivery of an agent in which the agent is introduced to the body across a mucous membrane which allows for the avoidance of the gastrointestinal tract and first pass liver metabolism and consequently allows the agent to directly enter circulation. This can include passage through the gastrointestinal tract as by oral ingestion, but refers to delivery through the mucosa of such locus.
As used herein, a chronic condition is one that is expected, based on physician’s experience and knowledge in the art to last more than 6 months, and generally more than a year. It is a condition that requires treatment for at least 6 months, and can require at 1 year or more, including for life.
As used herein, treating a subject with a disease, disorder, or condition means that a drug, therapeutic, composition, combination or other therapy is administered to the subject and the subject’s symptoms or manifestations of the disease or conditions are partially or totally ameliorated, or remain static (do not worsen) following treatment. In examples, treating a subject with a disease, disorder, or condition includes treating a subject with a rotational combinatorial therapy described herein.
As used herein, a pathway refers to a biological pathway that is involved in or mediates disease, disorder, or condition. A pathway can be targeted via a component of the pathway, such as a receptor or ligand involved in the pathway. Depending upon the disease, disorder, or condition and the role of the pathway in the disease, disorder, or condition, the targets can be antagonized or agonized.
As used herein, a “combination” refers to any association between two or among more items. The association can be spatial or refer to the use of the two or more items for a common purpose. A combination for therapeutic use(s), such as a rotational combinatorial therapy provided herein, includes more than one therapeutic (z.e., medication), such as two, three, four, five, or more therapeutics, but generally includes up to five therapeutics in the combination. The rotational combinatorial therapy provided herein includes a regimen in which two, three, four, five or more combinations are rotated. The combinations for rotation in the regimen provided herein can contain different therapeutics or can contain some of the same therapeutics, but do not contain all of the same therapeutics in the combinations. A “combination” is used herein interchangeably with a cluster, which includes two or more treatments, such as two or more medications. Each combination can be administered as a therapy, or can be rotated and administered for a predetermined time, which can be shortened or lengthened according to the judgement of a physician. When not rotated each combination generally includes at least 3 different treatments
As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Hence, treatment encompasses prophylaxis, therapy and/or cure. Treatment also encompasses any pharmaceutical use of the compositions and combinations herein. Treatment also encompasses any pharmaceutical use of a combination of pharmaceutical and non-pharmaceutical therapeutics and compositions provided herein. Treatment encompasses a rotational combinatorial therapy described herein, in which a combination of therapeutics is administered in accord with any regimen described herein.
As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic(s), refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic(s). In some examples, amelioration of symptoms includes amelioration of side effects of the composition or therapeutic(s). In general, amelioration of adverse symptoms of a disease or disorder is a decrease in number or severity of the symptoms of the disease compared to the number or severity of the symptoms of the disease prior to treatment. In some examples, amelioration of a disease or condition can include a decrease in levels of a biomarker of the disease, such that the biomarker level is decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the biomarker level prior to treatment. In other examples, symptoms of the disease are decreased, such as the amount or severity of symptoms that are decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the amount or severity of symptoms level prior to treatment.
As used herein, incidence refers to how often an event occurs. For example, adverse side effect incidence refers to how often adverse side effects occur after administration of a therapeutic, for example the frequency of adverse side effects after administration of a combination therapy provided herein. For example, an adverse side effect(s) can occur 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the time after administration of a rotational combinatorial therapy provided herein. In examples herein, adverse side effects incidence decreases when multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein compared to a monotherapy or compared to a combination that is not rotated (z.e., is continuously administered or is administered over a longer period of time). For example, the incidence of an adverse side effect(s) can decrease 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% multiple therapeutics are formulated in a combination and administered in accord with a rotational regimen provided herein, compared to a monotherapy or compared to a combination that is not rotated.
As used herein, severity refers to degree of illness or symptoms of the illness or side effects of the illness or side effects of treatment s) for the illness manifested by a patient. The skilled physician can assess the severity of an illness or progression of the severity of an illness or the severity of side effects of the illness or side effects of treatments with therapeutics for the illness based on the knowledge in the field of medicine and pharmacology. The skilled physician or scientist can assess the adverse side effects associated with administration of therapeutics for a disease, disorder, or condition; for example, as severe e.g., adverse side effects requiring medical care), or as mild (e.g., adverse side effects not requiring medical intervention or treatment). For example, adverse side effects can be classified by severity using medical diagnosis codes used in clinical assessment and tracking, such as classification of severity as minor, moderate, major, and extreme. In some examples herein, adverse side effects following administration of the rotational combinatorial therapy provided herein are less severe than adverse side effects following administration of monotherapy or following administration of a combination therapy that is not rotated.
As used herein, “adverse effect” or “adverse side effect” refers to a harmful, deleterious and/or undesired effect of administering a medication or drug. Side effects or adverse effects are graded based on toxicity and various toxicity scales exist providing definitions for each grade. Exemplary of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Generally, the scale is as follows: Grade 1 = mild side effects; Grade 2= moderate side effects; Grade 3= Severe side effects; Grade 4= Life Threatening or Disabling side-effects; Grade 5= Fatal. Assigning grades of severity is within the experience of a physician or other health care professional.
As used herein, a dose-limiting toxicity (DLT) refers to the dose of a drug that produces side effects severe enough to prevent larger doses being given. It is within the level of skill of a skilled physician to assign or determine a DLT depending on the treatment protocol, the administered treatment, the disease to be treated, the dosage regime, and the particular patient to be treated. Generally, for the treatments, protocols, and regimens provided herein, a DLT is the dose of a drug results in an adverse event or side effect that on the toxicity scale of at least an ongoing or persistent Grade 2 toxicity that fails to resolve over the course of treatment and that limits the patient’s ability to comply with the protocol therapy. As part of rotational therapy, a drug that results in such side effects can be administered at a lower dose, since the combinations of drugs/treatments can act synergistically, or the drug can be discontinued, and optionally replaced with another drug with similar activity or effect.
As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced. Prophylaxis includes reduction in the risk of developing a disease or condition and/or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease and/or a prevention of worsening of symptoms or progression of a disease or reduction in the risk of worsening of symptoms or progression of a disease. Prevention includes inhibition or avoidance of a disease, disorder, or condition by administration of a rotational combination therapy provided herein.
As used herein, a “prophylactically effective amount” or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound, which, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses, such as a rotation of combinations of therapeutics. Thus, a prophylactically effective amount can be administered in one or more administrations. A prophylactically effective amount of a combination therapy as described herein can be lower than a prophylactically effective amount of a monotherapy. A prophylactically effective amount of a rotational combinatorial therapy as described herein can be lower than a prophylactically effective amount of a monotherapy or a combination therapy that is not rotated.
As used herein, an “effective amount” of a compound or composition for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount can be administered as a single dosage or can be administered according to a regimen, whereby it is effective. The amount can cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. A measure of effective amount or dosage is the median effective dose (ED50), which is the dose that produces a response that is fifty percent of the maximum obtainable response. As described herein, an effective amount of an individual therapeutic or a therapeutic combination is the amount required to achieve a desired amelioration of symptoms. A rotational combination therapy as can employ a lower effective amount of drug compared to a than a monotherapy or a combination therapy that is not rotated.
As used herein, a therapeutically effective amount or a therapeutically effective dose refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting, or partially arresting a symptom of a disease or disorder. A therapeutically effective amount or dose can reference the amount or dose of a particular therapeutic in a combination, and/or can reference the amount or dose of the combination of therapeutics in a combination.
As used herein, “therapeutic efficacy” refers to the ability of an agent, compound, material, composition, or combination of agents, compounds, materials, or compositions containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, composition, or combination of agents, compounds, materials or compositions containing a compound has been administered. For example, the therapeutic efficacy can refer to the therapeutic efficacy of a combination of therapeutics or a plurality of combinations, such as the combination(s) for use in a rotational combinatorial therapy.
As used herein, a standard dosage of a therapeutic is the dosage for formulation or administration that is approved by applicable regulatory agencies in the field, for example the U.S. Food and Drug Administration (FDA), such as the FDA Data Standards Advisory Board. In some examples a standard dosage includes an amount (z.e., mass) of a therapeutic (ie., drug) for formulation. A standard dosage also can include a dosage regimen for administering the standard dosage (z.e., biweekly dosing). A standard dosage as approved by a regulatory agency or body also can be a range of dosages or the dosage can vary based on characteristics of the patient to whom the therapeutic is administered. For example, the dosage can vary depending on the weight of the patient, or the severity of the disease or condition for which the therapeutic(s) is/are administered. A standard dosage also can be defined by pooled analysis using scientific studies of real-world data, where efficacy is established for various doses, and the dosage is established as a dosage with a particular efficacy. When standard dosage is established, efficacy, adverse side effects, and other factors are considered. An individual therapeutic (ie., drug) in a combination for a rotational combinatorial therapy provided herein can be formulated in a standard dosage. In some examples, an individual therapeutic (z.e., drug) in a combination for a rotational combinatorial therapy provided herein is initially formulated in a standard dosage and the dosage is modified from the standard dosage for subsequent administrations. For example, the dosage of the individual therapeutic is formulated in a lower or higher dosage compared to a standard dosage.
As used herein, polypeptide, peptide, and protein refer to polymers of amino acids of any length. Where not used interchangeably other characteristics, such as size, and structure, are contemplated, as defined below. The polymer can be linear or branched, and can contain amino acids, including modified amino acids, and it can be interrupted by non-amino acids. Also included are amino acid polymers that include sequence modifications including, replacements, insertions, deletions, and transpositions. Also included are amino acid polymers that contain post-translational modifications, such as disulfide bonds, glycosylation, sialylation, conjugation to other proteins, peptides, and polypeptides, such, but not limited to, conjugation to a detectable marker, or reporter.
As used herein the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, and amino acid analogs and peptidomimetics.
As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224). Such substitutions can be made in accordance with those set forth as follows:
Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions. Amino acid substitutions contemplated include conservative substitutions, such as those set forth in the table below. Substitutions that alter properties of the peptides cam be effected; such substitutions are generally non-conservative, but can be readily effected by those of skill in the art. Conservative amino acid substitutions generally can be effected without altering the activity of a peptide. Variants of the weight loss peptides are known and/or can be used herein.
As used herein, a protein is a polypeptide that has a three-dimensional structure and can include bonds in addition to peptide bonds, such as disulfide bonds and other interactions, that participate in forming the two- and three-dimensional structure.
As used herein, a peptide refers to a polypeptide that is from 2 to 300, but generally shorter than 100, amino acids in length. Peptides include therapeutic peptides that are administered for treatment of a disease, disorder, or condition. Peptides that are administered for therapeutic treatment are administered in an amount effective to elicit a therapeutic effect. Peptides for treatment can be administered at a dosage wherein the resulting circulating or accumulating levels of the peptide mimic normal levels or the peptide levels of a person who is not afflicted by the disease or condition. A therapeutic peptide or peptides also can be administered at a dosage so that the circulating level of the peptide is similar or the same of an endogenous peptide that elicits a therapeutic effect. For example, as described herein, gastric bypass is the most effective treatment to effect sustained and significant weight loss. It is known in the art that gastric bypass, not only reduces the size of the stomach to thereby limit food intake; it also results in changes in peptide hormones involved in regulating food intake and effects (see, e.g., Beckman et al. (2010) J Am Diet Assoc. 110:571-584, doi: 10.1016/j.jada.2009.12.023). In accord with the combinatorial therapeutic methods and regimens provided herein, peptides (or agonists), such as glucagon-like peptide-1 (GLP-1) (SEQ ID NO: 1), peptide tyrosine-tyrosine (PYY) (SEQ ID NO:6), and leptin (SEQ ID NO:3) that increase after gastric bypass can be exogenously administered to overweight or obese patients to mimic peptide levels and patterns of expression observed after gastric bypass to effect weight loss. Antagonists of peptides that decrease after bypass can be administered.
As used herein, a polypeptide is an amino acid chain that contains a plurality of peptides, and is generally 100 amino acids or longer. For purposes herein, polypeptides and peptides can be used interchangeably to refer to the therapeutics, such as those for weight loss.
As used herein, parenteral and parenterally refer to administration of an agent via any route other than oral administration. Parenteral includes the injection of a dosage form into the body by a sterile syringe or some other mechanical device, such as, for example an infusion pump. For example, parenteral administration includes injection (z.e., subcutaneous, intramuscular, and/or intravenous injection), infusion, implantation, intraperitoneal routes of administration, and any other mode of delivery other than ingestion to any site in or on the body of a subject.
As used herein, monotherapy refers to the use of a single therapeutic (z.e., drug or medication) to treat a particular disorder or disease. A monotherapy can include continued treatment, such as for months or years, with a single therapeutic.
As used herein, combination therapy refers to the administration of two or more different therapeutics or other treatment(s), such as radiation and surgery. Multiple therapeutic agents or treatments in the combination therapy can be provided and/or administered separately, sequentially, intermittently, simultaneously, or provided in a single composition. Generally, the two or more different therapeutics or other treatment(s) in a combination therapy are administered together or separately, or intermittently. For example, the treatments can be within 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, or 1 week of the other treatments in the combination. The timing and order of administration depends upon the disease, disorder, or condition, and is within the judgement of a physician.
As used herein, an orexigenic pathway is one that promotes appetite. Orexigenic pathways can be activated by increased AMP-activated protein kinase (AMPK) activity by ghrelin to promote appetite (orexia). The peptides orexin A (SEQ ID NO:36), neuropeptide Y (NPY) (SEQ ID NO: 35) and ghrelin (SEQ ID NO:23) stimulate appetite and act through orexigenic pathway(s).
As used herein, a receptor is a molecule that has an affinity for a particular ligand. For example, a receptor is a protein that specifically binds a signal molecule and then initiates a response. Receptors can be naturally-occurring or synthetic molecules. Receptors also can be referred to in the art as anti-ligands. In some examples, multiple receptor types are involved in a signaling pathway. In the context of pharmacology, receptors include macromolecules in the membrane or inside the cell that specifically (chemically) bind a ligand (drug).
As used herein, downregulate or downregulation refers to reducing or suppressing the body’s response to specific stimuli. Receptor downregulation is characterized by a decrease in total receptor number in the cell. Receptor downregulation can be caused by long-term exposure to agonists. Cells undergo receptor downregulation as a reversible process of adaptation, or desensitization, whereby a prolonged exposure to a stimulus decreases the cellular response to that level of exposure. In response to continuous drug exposure, receptor downregulation occurs and can decrease the drug efficacy due. Rotational and/or combinatorial drug therapy described herein decreases receptor downregulation due to the activation of multiple pathways as opposed to the one activated pathways in monotherapy. Rotational and/or combinatorial drug therapy described herein also can overcome the decreased therapeutic response due to receptor downregulation by rotating to a next combination with therapeutics that activate a pathway that is not downregulated.
As used herein, body mass index (BMI) is a value derived from the mass (weight) and height of a person. The BMI is defined as the body mass divided by the square of the body height, and is expressed in units of kg/m2, resulting from mass in kilograms and height in meters. BMI value is used to categorize a person as underweight (under 18.5 kg/m2), normal weight (18.5 to 24.9), overweight (25 to 29.9), or obese (30 or more) based on tissue mass (muscle, fat, and bone) and height. BMI under 20 and over 25 have been associated with higher all-causes mortality, with the risk increasing with distance from the 20-25 range.
As used herein, weight loss means a decrease in body mass of a person. Weight loss includes overall loss of fat, muscle and water compared to a pre-set time point. In examples herein, weight loss is assessed after treatment with a rotational combinatorial therapy compared to weight prior to treatment.
As used herein, off label use means the U.S. Food and Drug Administration (FDA) has approved the drug for a particular use and the approved drug is used for a different therapeutic use. The FDA has determined the benefits of using the drug for a particular use outweigh the potential risks, but has not assessed the risk or benefits of the drug for the off-label use.
As used herein, potency is the drug concentration required to produce an effect of a specified intensity. Potency generally is calculated as the concentration (or dose) required to produce 50% of the drug’s maximal effect (EO50). EC50 is used to express the potency in in vitro studies and also is the dose required for an individual to experience 50% of the maximum effect, and median effective dose or ED50 is used to measure a drug’s potency in a population (z.e., animal studies or human populations). ED50 is the dose that produces the desired effect in 50% of the population. As applied to clinical settings, potency can indicate the dose of the drug, whereas efficacy can indicate the magnitude of the response (regardless of the dose).
As used herein, an “adverse effect,” or “side effect,” or “adverse event,” or “adverse side effect,” refers to a harmful, deleterious and/or undesired effect associated with administering a therapeutic agent. For example, side effects associated with the administration of a monotherapy, such as continuous administration of a monotherapy over an extended period of time. Such adverse side effects include, for example, headaches, nausea, diarrhea, heartburn, gas, constipation, dry mouth, dizziness, increased blood pressure, increased heart rate, restlessness, drug dependence, abuse, and withdrawal symptoms. Other serious adverse effects include infections, such as tuberculosis, and other infections caused by viruses, fungi and bacteria, including upper respiratory infections, as well as dermatological and dermal toxicity, such as rash. Thus, “adverse effect” or “side effect” refers to a harmful, deleterious and/or undesired effect of administering a therapeutic agent. Side effects or adverse effects are graded on toxicity, and various toxicity scales exist, providing definitions for each grade. Examples of such scales are toxicity scales of the National Cancer Institute Common Toxicity Criteria version 2.0, and the World Health Organization or Common Terminology Criteria for Adverse Events (CTCAE) scale. Assigning grades of severity is within the skill of an experienced physician or other health care professional. The severity of symptoms can be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) grading system. The CTCAE is a descriptive terminology used for Adverse Event (AE) reporting. The grading (severity) scale is provided for each AE term. The CTCAE displays Grades 1 through 5, with clinical descriptions for severity for each adverse event based on the following general guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Death related to AE/ fatal).
As used herein, serum level refers to the amount of a therapeutic in blood plasma. Serum level can be used to assess whether the amount of the therapeutic administered is safe and/or effective. Therapeutic drug monitoring of the concentration of mediation(s) in body fluids such as blood serum can be used during treatment and/or for diagnostic purposes. Assessing drug serum levels can be used, for example, to avoid drug toxicity, to determine if drug serum level is toxically high; to adjust dose, such as, for example, after reaching a steady state, to determine if the loading dose was adequate, and/or to predict a patient’s dosing requirements; and for monitoring patient compliance, diagnosing undertreatment (ie., when drug dose can be increased), and diagnosing ineffective treatment.
As used herein, comorbidity refers to the presence of one or more additional conditions co-occurring (that is, concomitant or concurrent) with a primary condition. Comorbidity indicates that the one or more conditions occur simultaneously with the primary condition or as a result of the primary condition. For example, hypertension can be a comorbidity of obesity, where a subject has hypertension as a result of the obese state. In some examples, complications of the primary condition and the comorbidity are the same or overlap.
As used herein, disease or disorder refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those that can be treated with a rotation of pharmaceutical agents. Diseases and disorders also include those where treatment of the disease or disorder is impaired due to downregulation of receptors or drug targets, leading to a decreased efficacy of the therapeutics. Of particular interest herein are those disorders where treatment is not effected due to resistance to the expected efficacy of the therapeutic molecule or drug or peptide.
As used herein, “chronic” is used to characterize the duration of a disease, disorder, or condition. For example, a chronic condition is a condition that is prolonged and requires ongoing medical intervention or limit activities of daily living or both. A chronic condition generally is a condition that lasts more than 3 months, 6 months, 1 year or more, or that is terminal. A chronic condition can persist when the affected individual receives treatment or therapeutics, and during the amelioration of signs or symptoms of the condition. Chronic conditions include chronic diseases or disorders. Chronic conditions can be characterized by one or more of etiology, duration, onset, recurrence/pattem, prognosis, sequelae, diagnosis, severity and prevalence (see e.g., O’Halloran et al., Family Practice, (2004) 21(4): 381-386).
As used herein, “obesity” refers to a condition in which the subject has abnormal or excessive fat accumulation, such as a body mass index of greater than 30. Obesity can be a health risk. Obesity can be caused by a multitude of factors, including genetic and environmental factors. Patients with obesity can have other comorbidities, such as, for example, high blood pressure, type 2 diabetes, cardiovascular disease, high cholesterol, and others.
As used herein, “overweight” refers to a condition in which the subject has extra fat accumulation or weight that is higher than typical, with a body mass index that is, for example, greater than 25. Overweight also can be assessed by other methods known in the art, such as by assessing waist and/or hip circumference, subcutaneous fat thickness, percent of fat or muscle compared to total body composition, overall body weight, and similar assessments. Other metrics can be used to determine whether a subject is overweight, such as a waist to hip ratio of greater than, for example, 1.0. Overweight subjects are at increased risk various comorbidities, including high blood pressure, high cholesterol, COPD, type II diabetes, and others. Overweight can be caused by a multitude of factors, including genetic and environmental factors.
As used herein, a “pharmacokinetic property” refers to a property related to the action of a drug or agent, such as a therapeutic peptide, in the body and in particular the rate at which drugs are absorbed, distributed, metabolized, and eliminated by the body. Pharmacokinetics can be assessed by various parameters. These include, but are not limited to, clearance, volume of distribution and serum half-life. Pharmacokinetic properties of peptides can be assessed using methods well known in the art, such as, for example, administering the peptide to a human or animal model and assessing the amount of the peptide in the body (e.g., in the bloodstream) at various time points. The various parameters, such as clearance, volume of distribution and serum half-life, are assessed using calculations well known in the art and described herein.
As used herein, “improved pharmacokinetic properties” refers to a desirable change in a pharmacokinetic property of a peptide or combination of peptides or combination of therapeutics, such as peptides in a pharmaceutical composition for administration with the timing or regimen set forth herein, compared to, for example, a peptide or therapeutic administered continuously or once. The change can be an increase or a decrease.
As used herein, “synergistic effect” or “synergy” or grammatical versions thereof refers to a larger therapeutic effect of the combined treatment compared to the effect predicted from the sum of each therapeutic alone. For example, when the action of one drug is increased when administered in the presence of another drug.
As used herein, plasma half-life (t 1/2) refers the elimination half-life of a peptide(s) or therapeutic(s) or combinations thereof or the time at which the plasma concentration of the administered peptide(s) or therapeutic(s) or combinations thereof has reached one half of its initial or maximal concentration following administration. Reference to plasma half-life includes plasma half-life during the a-, P-, and/or y- phase. Plasma half-life can be assessed using methods well known in the art. For example, assays in which a peptide(s) or therapeutic(s) or combinations thereof is administered to subjects can be performed, and the plasma half-life of the peptide(s) or therapeutic(s) or combinations thereof assessed by measuring the amount of the peptide(s) or therapeutic(s) or combinations thereof in the plasma at various time points. The t’AB, for example, is calculated as -ln2 divided by the negative slope during the terminal phase of the log-linear plot of the plasma concentration-versus- time curve. In some examples, the plasma half-life can aid the skilled artisan in determining when a secondary peptide or therapeutic or combinations thereof can be administered to a subject, such as, for example, in a rotational therapy regimen described herein.
As used herein, clearance refers to the removal of an agent, such as a peptide, from the body of a subject following administration. Clearance can be assessed using methods well known in the art. For example, assessment of peptide levels in blood or serum or another fluid from a patient administered a peptide or combination of peptides or therapeutics can be performed, and the clearance of the peptides from the body assessed by measuring the amount of the peptide(s) in the plasma at various time points and calculating the clearance as Dose / AUC 0-inf. In some examples, clearance of a peptide results in a decrease in the circulating peptide levels in serum compared to circulating levels directly or recently after peptide administration. The clearance of administered peptide(s) or therapeutic(s) or combinations thereof can result in a decrease in circulating levels by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to circulating levels directly or recently after peptide administration.
As used herein, the term assess or assesses and grammatical variations thereof, is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a pharmaceutical(s) or therapeutic(s) or phenotype(s), and of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect. For example, assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein on a subject for weight loss can be evaluated by a variety of metrics, including fat, weight, water, and muscle loss or gain. Assessing the effect of administration of a combination therapy provided in accord with a rotational regimen described herein also can be evaluated by measuring levels of the therapeutics or other molecules in the bloodstream. In some examples, a decrease in the amount of the therapeutic(s) such as, for example, a therapeutic peptide or combinations of peptides in the bloodstream or serum can indicate that another peptide(s) or therapeutic(s) or combinations thereof can be administered to the subject. For example, a secondary therapeutic(s) (z.e., peptide e.g., therapeutic peptide) or combinations thereof that is different from the initially administered therapeutic(s) ) (z.e., peptide(s) e.g., therapeutic peptide(s)) or combinations thereof can be administered when the concentration of the primary is decreased by at least or about or about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the level or amount of the therapeutic(s) directly after administration, where the level of amount of the therapeutic(s) directly after administration is assessed at least or at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or more after administration.
As used herein, the term “subject” refers to an animal, including a mammal, such as a human being. Subjects include human patients.
As used herein, a “patient” refers to a human subject. In some examples “patients” or “subjects” are humans who participated in a therapeutic regimen, such as a therapeutic regimen described in the examples herein. In some examples a “patient” is a human who has a chronic condition, disorder, or disease, such as overweight or obesity. Patients or subjects can be treated with the rotational combinatorial therapy herein.
As used herein, “animal” includes any animal, such as, but not limited to, primates including humans, gorillas, and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs; and other animals. Non-human animals exclude humans as the contemplated animal.
As used herein, a “control” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be a subject, such as a subject that is not treated with rotational combinatorial therapy provided herein, or is treated with a monotherapy, or combination therapy that is not rotated, or a placebo. A control also can be an internal control.
As used herein, a “composition” refers to any mixture of two or more products or compounds, for example, but not limited to, peptides, therapeutic molecules, agents, modulators, and regulators. A composition can be, for example, a solution, a suspension, an emulsion, a liquid, a powder, a paste, aqueous or non-aqueous formulations, and any combination thereof.
As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass peptides or drugs, or therapeutics and combinations thereof contained in articles of packaging.
As used herein, “fluid” refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
As used herein, a “kit” refers to a packaged combination, optionally including reagents and other products and/or components for practicing methods using the elements of the combination. For example, kits containing a combination of pharmaceuticals provided herein and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of a biological activity or property are provided. Kits optionally include instructions for use.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising “a therapeutic” includes combinations with one or a plurality of therapeutics.
As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence, “about 5 hours” means “about 5 hours” and also “5 hours.”
As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally included therapeutic means that the therapeutic is included or is not included.
B. OVERVIEW
Provided are delivery vehicles that display peptides for weight loss and anabolic peptides. Provided herein are delivery vehicles, such as liposomes, exosomes, extracellular vesicles, lipid nanoparticles (LPNs), for displaying or containing peptides for treating obesity and/or diabetes, and optionally containing small molecule drugs in or on the vehicles.
Exemplary proglucagon derived peptides are provided, and anabolic peptides and other peptides and drugs that that promotes muscle growth and/or prevents or inhibits muscle wasting are described. The delivery vehicles display at least one of the proglucagon peptides and a muscle wasting/promoting peptide or a peptide that inhibits muscle loss, such as a myostatin pathway inhibitor, or a plurality thereof. Alternatively, or in combination, the delivery vehicles display one the peptides and combinations of at least three different delivery vehicles are administered together, such as in a co-formulation, or administered sequentially, or simultaneously. Generally, the delivery vehicles are formulated for administration by injection, such as intravenous injection or subcutaneous injection. The delivery vehicles also can be formulated for mucosal administration via contacting with oral mucosa, intestinal mucosa, and/or intranasal mucosa.
The delivery vehicles, include, but are not limited to, liposomes, such as those exemplified herein, or any suitable liposome, or a lipid nanoparticle (LNP), or an extracellular vesicle, including an animal or plant extracellular exosome, such as a milk-derived, or a microalgal extracellular vesicle (see, International PCT Publication No. WO2023/144127; International PCT application No. WO2023/076418;and International PCT application No. WO2023/076418). The peptides drugs can be displayed on or contained in or both using any suitable delivery vehicle, including extracellular vesicles, exosomes, LPNs, and liposomes. Additionally, small molecule drugs, such as phentermine, can be combined with the delivery vehicle, such as introducing it into extracellular vehicle or fabricating them together. Thus, delivery vehicles are provided that deliver combinations of drugs to treat obesity and reduce or eliminate the concomitant muscle loss that accompanies weight loss by enhancing muscle growth.
Suitable peptides and combinations thereof and regimens are described herein and in commonly owned International PCT application No. PCT/US23/77508 (now subsequently published International PCT publication No. WO2024/091863). Hence, provided are combinatorial treatment protocols and rotational combinatorial treatment protocols in which diseases, disorders, and conditions are treated by targeting a plurality of molecules and/or pathways involved in the disease, disorder, or condition, to reduce or avoid desensitization to a particular treatment, and also can reduce toxicity and adverse side effects. The diseases, disorders, and conditions include obesity, diabetes, and associated diseases, disorders, and conditions, such as obesity comorbidities.
The combination of treatments can be rotated so that a subject is treated with different combinations of treatments for limited periods of time. Also provided are methods for developing such protocols, including selecting a disease, disorder, or condition for treatment with a combinatorial protocol and/or a rotational combinatorial protocol. These protocols and methods are exemplified herein with respect to obesity, and several other diseases, disorders, and conditions that are difficult to treat and that become resistant to treatment over time. In accord with the disclosure herein, the peptides and other drugs are displayed on or provided in delivery vehicles, such as liposomes and exosomes, and formulated for administration to a subject, such as by injection, including subcutaneous injection, such as by autoinjector.
Obesity is exemplar of a disease, disorder, or condition, as shown herein that can be treated with a combinatorial, particularly a rotational combinatorial treatment protocol. Obesity is a medical condition in which excess body fat has accumulated. The excess body fat has adverse effects on health (see, e.g., “Obesity and overweight Fact sheet N°311,” published by the World Health Organization (WHO) in January 2015. Retrieved 2 February 2016). Obesity increases the likelihood of various diseases and physical and mental conditions. For example, these increases are manifested in metabolic syndrome (Haslam etal., (2005) Lancet 366 (9492): 1197- 209), and include cardiovascular diseases, high blood pressure, high blood cholesterol, high triglyceride levels, diabetes mellitus type 2, obstructive sleep apnea, certain types of cancer, osteoarthritis, and depression (Haslam et al., (2005) Lancet 366 (9492): 1197— 209). Obesity reduces life expectancy. (Jura etal., (2016) Age (Dordr) 38(1):23; Peeters etal., Ann lnternMed (2003) 138(l):24-32).
These health complications either are directly caused by obesity or indirectly related to obesity through mechanisms sharing a common cause such as a poor diet, a sedentary lifestyle, and/or genetics. The strength of the link between obesity and specific conditions varies; one of the strongest is the link with type 2 diabetes; approximately 64% and 77% of cases of diabetes in men and women, respectively, can be attributed to excess body fat (Maggio et al., Endocrinol Metab Clin North Am. (2003) 32(4):805-22).
The biochemical link between the development of obesity and health consequences fall into two broad categories: those attributable to the effects of increased fat mass, such as osteoarthritis, obstructive sleep apnea, and social stigmatization, and those due to the increased number of fat cells, such as diabetes, cancer, cardiovascular disease, non-alcoholic fatty liver disease (Stenkula etal., Am J Physiol Regul Integr Comp Physiol. (2018) 315(2):R284-R295). Increases in body fat alter the body's response to insulin, potentially leading to insulin resistance. Increased fat also creates a proinflammatory state and a prothrombotic state (Kawai et al., Am J Physiol Cell Physiol. (2021) 320(3):C375-C391; Moghbeli et al., Adv Clin Chem (2021) 101 : 135-168; Samad et al., Blood(2013) 122(20):3415-22; Bovolini et al., IntJ Sports Med. (2021) 42(3): 199-214).
Combinatorial protocols involve the administration of a plurality of treatments, each of which targets different pathways involved in the disease, disorder, or condition. The combinatorial protocols involve combination of at least three different treatments, such as at least two peptides that promote or result in weight loss and one that prevents or reduces muscle loss associated with weight loss. The combinatorial protocol(s) can be integrated into a rotational combinatorial protocol in which different combinations of treatment are rotated. Rotational protocols can be selected where monotherapy or combination of treatments become less effective or require higher doses to be effective. Provided herein are exemplary rotational combinatorial protocols and methods of developing such protocols.
Provided herein is platform in which combinations of weight loss and associated drugs are provided in or displayed on a delivery vehicle, such as an exosome or extracellular vesicle or LPN. Liposomes are exemplary of such vehicles. The peptide drugs can be displayed on the vehicle or incorporated into it; the small molecule drugs can be incorporated or associated with the surface so that they can be delivered together. The stoichiometry of the amounts of each drug can be adjusted to deliver the proper dosage, adjust, and provide relative amounts or ratios of the drugs for dosing the proper amount of each. The platform provides the ability to adjust various parameters and deliver combinations of drugs for treating obesity and concomitantly avoiding or reducing muscle loss that accompanies weight loss. The can be provided in or on separate vehicles, which can be mixed before administration to provide combinations.
In particular embodiments, the delivery vehicles, such as liposomes, provide more than a plurality of combinations (combinatorial) of the peptides (such as 3 weight loss peptides and 1 anabolic) providing an array of combinations to rotate and/or select the best for treating a particular subject. For example, each of the unique combinations can be rotated, such as every three months to prevent drug desensitization and reduce or avoid adverse side effects. The peptide stoichiometry can be adjusted, such as by modifying the surface of the liposome to reflect a dominant population of the more efficacious peptide or to increase the dose of one or more relative to others. The size of the delivery vehicle can be adjusted or selected to affect the amount of drug(s) in or on the liposome and to control binding kinetics of the surface peptides.
Liposome are among the delivery vehicles. Liposomes and other vehicles provide several advanced pharmacokinetic features, including: a. protection of the peptides from endogenous peptidases in serum, thus extending half life b. a delivery system within the liposome itself, of which we will describe below.
As detailed herein, combinational, combinatorial and rotational therapies provide advantages compared to monotherapy. They permits treatment via multiple pathways or targets involved in obesity or other disease, disorder, or condition.
The delivery vehicles also can for fabricated or formulated to deliver small molecule drugs including, but are not limited to: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, in addition to the peptides. These are known drugs; dosages can be established when used in combination with the other drugs and peptides. The combinations, thus, include at least two or three known peptides for treatment of obesity and additionally one or more peptides (or other drugs) that reduce or eliminates muscle loss that accompanies weight loss. Generally, the drugs that reduce or eliminate muscle loss re muscle enhancing anabolic peptides, such as sermorelin, tesamorelin, IGF-1, or other growth hormone or peptide derived therefrom. Other drugs that inhibit the causes of muscle loss also are known, and, optionally, can be included in the combinations. These include myostatin pathway inhibitors, such as the antibody bimagrumab, which inhibit the activity of myostatin. Myostatin also is known as growth differentiation factor 8 or GDF-8; it is a member of the transforming growth factor-P (TGF-P) superfamily. Myostatin is a negative regulator of skeletal muscle growth, so its inhibition can contribute preservation of lean muscle mass. Inhibitors of myostatin, thus, are distinct from muscle enhancers promote muscle growth; they have a different target and mode of action. Myostatin inhibitors are not muscle enhancers. It has been reported that myostatin pathway inhibitors used in combination with a GLP-1 pathway agonist can enhance overall weight loss (see, e.g., US patent application publication US 20240368262).
Advantages of the delivery vehicles, such as liposomes, provided herein include, but are not limited to:
1. Synergistic weight loss to the peptides (should, for example, exceed Tirzapeptide’s claimed efficacy of over 20% in 12 months).
2. Numerous combinations can be made with these molecules, in addition to surface PDP’s.
3. Resistance to drug sensitization.
4. Safety and profiles and dosing are known.
5. The delivery vehicles, such as the liposomes, can provide “dual peak” activity. They display peptides, which can have an immediate pharmacological effect, and then, once the vehicle, such as the liposome ruptures, there is a second time release of any weight loss push. There can be a further peak or release of any drug/peptide from the ruptured vehicle .
6. Since these are well-known drugs, serum levels and other parameters can be calculated easily from known prescribing doses (for example, phentermine is prescribed at 37.5mg daily) and then packaged into each vehicle in an amount to mimic that to achieve therapeutic serum levels and match other parameters.
7. The delivery vehicles can concurrently deliver the small molecule drugs to treat other diseases, such as diabetes (metformin), hypertension (ACEi/ARBS), dyslipidemia (Statins), and others for obesity co-morbidities.
Depending on the selected delivery vehicle, they can be administered by various routes, including subcutaneous injection, and oral, inhalation, and intramuscular. As described herein and known in the art, auto-injectors for selfadministration are routinely used for administration. Volumes up to 3 mL generally are administered. Higher volumes can be administered. For example, high speed autoinjectors can deliver higher doses, as can adding excipients, such as a hyaluronidase, to the compositions. The high speed autoinjectors inject fairly large volumes quickly, and the formulations also contain excipients, such as a soluble hyaluronidase, to facilitate administration of large volumes Typical GLP-l/GIP injectors, for example, deliver from 0.5ml to 3ml.
As described herein, small molecule drugs, such as phentermine can be incorporated into or onto the liposomes. Small molecules inside the liposome or other vehicle provide unique pharmacokinetic properties. For example, for liposomes exemplified herein, the peptides act immediately since they are on the outside of the liposome and actuate a chain of molecular events for weight loss synergistically (First phase). As the liposome dissolves, there are some small molecules inside the liposome that are then released (2nd Phase), and eventually any small molecules attached to the inner surface of the liposome will be available for binding to its receptor (third phase).
In exemplary embodiments, the vehicles are liposomes or exosomes that contain at least 2, generally at least three, peptide drugs, and additionally muscle enhancer, such as an anabolic hormone. Among the possible peptides and drugs, there are at least 12 different peptides, which are known to produce or facilitate weight loss, and at 2 or 3 anabolic peptides, exemplified herein. These include, for example: GLP- 1 (SEQ ID NO: 1), Leptin (SEQ ID NO:3), Oxyntomodulin (OXM; SEQ ID NO:4); PYY (SEQ ID NO: 6), Amylin (SEQ ID NO: 7); Tesamorelin (SEQ ID NO: 8), GIP, SEQ ID NO: 10; CCK (SEQ ID NO: 11); Glucagon (SEQ ID NO:27); Sermorelin (SEQ ID NO:5); and IGF1 (SEQ ID NO:44), and variants or biologically active portions of each as well known in the art and/or detailed herein. Other known peptides and small molecule weight loss drugs can be included.
As noted above, there are a variety of peptides and small molecule drugs that can be combined. There are more than 200 different combinations based on the lists herein. For example, among these are the following exemplary combinations:
Drugl : GLPl/GIPl/Oxyntomodulin + GH (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 4 or 47 + SEQ ID Nos: 5, 8, 44
Drug2 : GLPl/GIPl/Amylin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 7 + SEQ ID Nos: 5, 8, 44
Drug3: GLP1/GIP1 /Glucagon + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 27 + SEQ ID Nos: 5, 8, 44
Drug4: GLP1/GIP1/CCK+ GH (Sermorelin or Tesamorelin or IGF1)
SEQ ID Nos: 1, 10, 11 + SEQ ID Nos: 5, 8, 44
Drug5: GLP1/GIP1/PYY + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 6 or 37 + SEQ ID Nos: 5, 8, 44
Drug6: GLP1/GIP1 /Leptin + GH (Sermorelin or Tesamorelin or IGF 1) SEQ ID Nos: 1, 10, 3 + SEQ ID Nos: 5, 8, 44.
For displaying on delivery vehicles, the polypeptides can be pegylated or modified by group, such azido for click chemistry. Delivery vehicles are prepared by known methods, including those exemplified herein, for linkage of the polypeptides. In some embodiments, Lysine (K) residues, for which pegylation reduces or eliminate activity of the peptide when displayed, are replaced with a conservative amino acid replacement, such as Arg, Glu, Gin, His, so that only a lysine or lysine residues that do not substantially alter activity are available for pegylation. For example, Peptides can be linked to pre-formed liposomes or linked to components of the liposomes during preparation of the liposomes. A goal is to preserve as much of the tertiary conformation of the native peptides need to retain activity, reduce steric hinderance between or among the up to 4 peptides on a single liposome, protect the N-terminus from serum proteases, and/or, for example, set up an azio-PEG24 linker for copper- catalyzed azide-alkyne cycloaddition (CuAAC) and bioconjugation to preformed liposomes.
The stoichiometry can be assessed, for example, in vitro via cAMP assays as described below. As an example, liposomes are fabricated as exemplified. The peptides can be acetylated at the N-terminus to protect them from in vivo degradation. The activity of the peptides displayed on the resulting delivery vehicles can be assessed by known assays, including commercially available kits. These kits are based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenous labeled cAMP conjugate that acts as a competitor. This is followed by detection of the labeled cAMP conjugate using a variety of detection technologies, including fluorescence resonance energy transfer (FRET) or enzymatic reactions. Based on known properties of our quad-peptide liposome, each activating cAMP messenger pathway, we expect a significant increase in intracellular cAMP over baseline and single, double, or triple peptide liposome. Adipose-derived stem cells (ADSCs) cell line can be used with any of these kits for assaying in vivo cAMP. Several kits available on the market that can be used to measure intracellular cAMP levels, including, for example:
1. HTRF cAMP kit from Cisbio,
2. LANCE cAMP kit from PerkinElmer,
3. HitHunter cAMP kit, 4 DiscoverX cAMP kit,
5. Abeam and BioVision cAMP Direct Immunoassay Kit,
6. GloSensor cAMP assay from Promega.
C. Delivery vehicle -components
The peptides are provided linked to or incorporated into the surface of delivery vehicle. The following sections describe that the vehicles contain three components or aspects: 1) the delivery vehicle; 2) linkers or linkages; 3) the peptides. Peptides listed herein can be incorporated in and/or on the delivery vehicles.
1. Component -Proglucagon derived Peptides (PGDP) and muscle enhancement peptides
In obesity, several organs are involved in the orchestration of body weight homeostasis. These include the pancreas (a-cells of the islets of Langerhans), gut (intestinal enteroendocrine L-cells), and brain (caudal brainstem and hypothalamus). The proglucagon gene is expressed in these organs and produces several key hormones that regulate satiety; these hormones are referred to as proglucagon-derived peptide (PGDP). Bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin and glicentin-related pancreatic peptide, which are produced via tissue-specific processing of proglucagon by the prohormone convertase (PC) enzymes, PC 1/3 and PC2 (Figure 1). PGDP peptides exert unique physiological effects that influence metabolism and energy regulation; several of these peptides have been exploited in the form of long-acting, enzymatically resistant analogues for treatment of various pathologies. These can be linked to delivery vehicles (or incorporated in the surface thereof) as described herein for use in combination with other such peptides and/or delivery vehicles. a. Proglucagon Derived Peptides and tissue specific secretion
Proglucagon is expressed in both alpha-cells of the pancreatic islets as well as neuroendocrine L-cells primarily located in the distal ileum and colon. However, the PGDP profile is not identical in the pancreas and gut, due to differential post- translational processing of proglucagon by tissue-specific enzymes termed prohormone convertases (PC). It is accepted that pancreatic alpha-cells mainly possess PC2, which cleaves proglucagon to generate glicentin-related pancreatic peptide (GRPP), glucagon, intervening peptide- 1 (IP-1) and major proglucagon fragment (MPGF). In contrast, in the L-cell, proglucagon is cleaved by PC 1/3 yielding glicentin, GRPP, oxyntomodulin (OXM), GLP-1, intervening peptide-2 (IP- 2) and GLP-2. Some degree of crossover exists.
The gut and brain are extra-pancreatic sources of glucagon, while local intraislet GLP-1 production has also been established in alpha cells, particularly in times of beta-cell stress. In the brain the solitary nucleus of the medulla oblongata which utilizes PC 1/3 in a similar fashion to the gut generates PGDP’s in the central nervous system (CNS) b. Unimolecular multi-agonists
Employing combinations of single gut hormones or analogues provided a sound basis for the application of multi-agonism in T2DM. With the combination of liraglutide plus an acylated GIP analogue, synergy was demonstrated leading to improved glucose-lowering and insulinotropic actions in obese-diabetic mice compared to either of the individual incretin analogues alone. Based on this, we conceive that unimolecular multi-agonists represent the next step in future in the therapeutic application of PGDPs, with increasingly complex and experimental molecules being developed. This is evidenced by the observation that secretion and action of a number of gut hormones, including the PGDPs GLP-1, GLP-2, OXM and glicentin, together with PYY, GIP, cholecystokinin (CCK), neurotensin (NT) and secretin, are positively modulated in concert following Roux-en-Y gastric bypass (RYGBP or RYGB). These are major determinants in the improvements of appetite, body weight, glucose tolerance and insulin sensitivity demonstrated post-surgery. In view of the costs, limited availability, risks associated with surgical procedures, methods and regimens and products provided herein are designed to emulate the post- surgical, hormonal mechanisms of RYGB. The combination therapies provided herein can evoke an array of positive actions within various organs, thereby surpassing advantages observed with individual peptides. c. Enhancing fat loss efficacy via proglucagon derived peptides (PGDP) and co-secretory molecules released after a RYGBP
Adipose tissue plays a central role in various disease states such as metabolic syndrome, diabetes, coronary' artery disease etc. Focusing on fat loss ignores skeletal muscle, another relevant tissue that affects general health, skeletal muscle (SM) is the body’s largest non-fat component and can contribute up to 40% of the adult human body weight and be responsible for 30% of energy expenditure. It is also an established independent predictor of cardiometabolic diseases and mortality. Previous studies have reported that low levels of muscle mass are associated with an increased risk of insulin resistance, and CVD. Low muscle-to-fat ratios were associated with an increased risk of metabolic syndrome, diabetes, and cardiovascul r mortality.
Based on peptide changes after a RYGBP, and the concept of enhancing muscle-to-fat ratio, we propose a system that can increase fat loss and enhance muscle mass using liposomes or exosomes with at least one or more peptides for fat loss, and 1 or more peptides for muscle enhancement. We also consider overcoming the pharmacological phenomena of tolerance and desensitization to chronic exposure to the same drug by adjusting the combination of the fat loss (FL) and (ME) peptides and rotating them over intervals of 3 months. This multi-agonist approach uniquely combined and rotated would maintain weight loss over longer periods as compared to monotherapy or combined therapy without rotation of peptides. As detailed in the disclosure herein, a goal is to provide regimens that mimic secretory and pulsatile response of these endogenous peptides that occur following RYGBP and other bariatric weight loss surgeries. Provided are delivery vehicles for effecting and providing the combination therapies. Combinations of peptides are described throughout the disclosure herein. It is to be understood that the mixtures of peptide therapeutics can be linked to or incorporated into a delivery vehicle, such as a liposome or extracellular vehicle, such as an exosome, or a synthetic lipid-based nanoparticle. The delivery vehicles can be prepared to deliver a single peptide or a plurality thereof, mixed to provide complementary activities to mimic hormonal and other changes that occur following bariatric surgery. The general approach is to administer one or two peptides for fat/weight loss and one or more to prevent or inhibit muscle loss associated with fat loss or weight loss, or a peptide that promotes muscle growth. For example, two or three proglucagon peptides can be linked to a liposome, and a peptide that inhibits or prevents muscle wasting or that enhances muscle production can be prepared. Alternatively, few than all of the peptides can be linked to a liposome, and mixtures of liposomes delivering the combination of peptides can be co-formulated or administered at the same time or serially. Included among the peptides for linkage to or conjugation to or incorporation into a delivery vehicle, such as a liposome, are the following:
Peptides for fat loss include (FL=fat loss):
PeptideFLl=GLP-l
PeptideFL2=Adiponectin
PeptideFL3=Leptin
PeptideFL4= Oxyntomodulin
PeptideFL5=PYY
PeptideFL6= Amylin
PeptideFL7=Pancreatic peptide
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide)
PeptideFL9= Glicentin PeptideFLIO = Glucagon
PeptideFLl 1=GRPP
PeptideFLl 2=HGH 176-191
Peptide FL13= CCK
PeptideFLl 4= Neurotensin
PeptideFLl 5= Secretin
PeptideFLl 6= IP1
PeptideFLl 7= MPGF (major proglucagon fragment)
Peptides for muscle enhancement ME:
PeptideMEl=Sermorelin
PeptideME2=Tesamorelin
PeptideME2=IGFL
2. Delivery vehicles
Delivery vehicles include any lipid nanoparticles on which peptides can be displayed on the surface and that are biologically compatible so that they can be administered to a human subject. Delivery vehicles include, but are not limited to, lipid nanoparticles, extracellular vesicles, liposomes, and other such delivery vehicles known to those of skill in the art. Exemplary of lipid-based delivery vehicles are exosomes, such as extracellular delivery vehicles, and liposomes. The peptides can be incorporated into the surface of the vehicles, such as by recombinant expression in a surface protein, or by chemical linkage to a moiety on the surface. The surface can be modified for linking peptides by methods known to those of skill in the art, including those described herein.
Liposomes are exemplary delivery vehicles
Liposomes are versatile drug delivery vehicles. They have unique properties, which include, for example, site-targeting, sustained or controlled release, protection of drugs from degradation and clearance, superior therapeutic effects, and lower toxic side effects. Liposomes include large multilamellar vesicles (LMV), and small unilamellar vesicles (SUV). Lamellas are phospholipid bilayers, in which the fatty acids are inside the bilayer, and polar head groups are outside in contact with water. LMVs can have diameters in micrometer range, while SUVs can be classified as nanoparticles, because diameter is typically under 100 nm. SUVs can traffic in vivo in the blood and go anywhere blood goes.
Liposome preparation include any of the following techniques, assuming the smallest possible particle to effectively carry at least 3 or 4 total (2 o3 fat loss (FL) peptides, 1 muscle enhancing (ME) peptide peptide) peptides without causing steric hinderance for each of their respective targets. Film hydration methods, double emulsification method, solvent injection techniques, sonification, and in situ preparation of liposomes are well-established methods for preparing the liposome.
To ensure stability and for performance of the liposomes, size reduction techniques as briefly described are employed. This includes, for example, (ultra)sonication either by bath or probe, French press Barenholtz, extrusion, homogenization, or combination methods, such as freeze-thaw extrusion, freeze-thaw sonication, and a high-pressure homogenization-extrusion technique [Pupo], Among these techniques, extrusion, and high-pressure homogenization (HPH) are among the most frequently employed in pharmaceutical manufacturing.
Liposomes can be prepared using phospholipids, such as phosphatidyl choline (PC), ethanol amine (PE), and serine (PS), or phosphatidic acid (PA). Phospholipids include those of natural origin, such as egg yolk, and also cholesterol, such as synthetic cholesterol, Cholesterol, which makes the bilayer tighter and less permeable, can be added. The molar percentage of cholesterol should be less than 60%, 50%, 40% or less. To prepare liposomes, the phospholipids, and other components first are dissolved into a volatile solvent, such as chloroform, methanol, t-butanol, cyclohexane, or a mixture thereof, so that components are evenly distributed. The Concentration of the phospholipids and other component can be low, such as less than 30 mg/ml, less than or equal to 20 mg/ml, less than 15 mg/ml or lower. Solvent then is evaporated, for example, by a rotary evaporator. For in vivo applications the solvent should be completely removed by keeping the lipid mixture under high vacuum for several hours. Dried lipid mixture is hydrated, such as with physiological salt solution (0.9 % NaCl), or other suitable buffer. Hydration can take several hours. At this point phospholipids are in thick multilamellar film or cake. Mechanical mixing leads to the formation of the LMVs. More powerful methods such as sonification, or high pressure microfluidic spraying break LMVs into SUVs. If hydration solution contains some drug molecules they are partially incorporated inside liposomes. For purposes herein, the biologically active peptides are conjugated to the outer surface of the liposomes.
3. Conjugation/Binding of peptides on the liposomes
Method for linking peptides to liposomes are well known (see, e.g., Frisch et a/.(1996) Synthesis of short polyoxyethelene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes, Bioconjugate Chem. 7: 180). Any of several methods can be used to attach peptides and proteins to the liposomes. First liposomes that are prepared that some lipids that have a reactive group that can be used for the coupling reaction. Exemplary groups include, but are not limited to, amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p-nitrophenyl carbonate, azide, and alkyne. Amino groups form amide bond with an activated carboxylic ester, such as N-hydroxy succinimide (NHS; see. e.g., Redford etal., (1991) Cholesterylsuccinyl-N-hydroxysuccinimide as a cross linking agent for the attachment of protein to liposomes, Biochem Pharmacol. 77:307)). Thiols bind with maleimide, bromo- or iodo acetyl, pyridyldithio groups. Amines form an amide bond with carboxylate in the presence of water soluble carbodiimide (EDS), and N-hydroxy succinimide (NHS).
Most amino couplings involve the lysine amino group but coupling also can occur at the terminal amino group. If one of these amino groups is at the active binding site, conjugation, such as PEG conjugation leads to reduction of the activity. Hence, for purposes herein, pegylation is effect whereby lysines in the active are not pegylated. A method for preparing peptides in which selected lysines are pegylated is described below. This is effected by PEGylating the lysine residue prior to synthesis and employing the pegylated residue during peptide synthesis. This method can be used in general for preparing pegylated peptides and polypeptides.
Other methods include modification of the peptides to replace lysine with a conservative residue and then PEGylating the modified polypeptide, selecting amounts of PEG moiety so that not all lysines residues are pegylated. For peptides in which the lysines are not accessible or not in an active site or such that pegylation does not affect secondary and tertiary structure, the peptides are pegylated by standard methods.
Hydrazides bind with carbonyl groups. P-Nitrocarbonate reacts with amines forming an amide bond. Azide and alkyne groups bind with each other very fast in the presence of a copper ion catalyst (click chemistry). Site specific attachment of PEG can be achieved using a transglutaminase enzyme to couple succinimide activated PEG to glutamine.
4. Spacers
Because of the diversity of the size of peptides including a linker is a strategy for loading a drug, such as a polypeptides on the liposome. Spacers can be included between the liposome, and peptide. Spacers allow peptides to orientate better for the binding with the receptor. Exemplary of spacers are water soluble polymer chains. For example, polyethylene glycol (PEG) is a commonly used spacer. Oligonucleotides also can be used as spacers. Oligonucleotides allow specific binding of selected peptides so that the amount of peptides can be better controlled. To employ oligonucleotides as spacers, first, selected oligonucleotides are bound with liposomes. Typically, oligonucleotides are bound in the same ratio as they are in the reaction mixture. The actual ratio of oligonucleotides can be measured by using a set of complementary oligonucleotides that are conjugated with fluorescent labels. If the ratio of oligonucleotides on the liposomes is equal to the desired ratio, the liposomes can be used as such. If correction is need, some additional oligonucleotides can be used. If there is a deficiency of some specific oligonucleotide, one or more of these additional oligonucleotides can be used to supplement this deficiency Peptides are conjugated with complementary oligonucleotides and mixed with the liposomes in a hybridization buffer, whereby that peptides are bound in a desired ratio.
Somewhat different methods of attaching peptides on the liposomes employ biotin-streptavidin binding. One streptavidin has four biotin binding sites. One, two, or even three can be used to bind streptavidin with a liposome, and at least one is still available for the binding of peptides. PEG spacers can again be used. On one end is attached biotin, and the other end can be NHS active ester that easily forms an amide bond with PE, when pH is right (slightly basic). The liposomes can be coated with a monolayer of streptavidin. Peptides similarly can be functionalized with biotin-PEG- NHS. The final product is obtained by mixing these peptide derivatives with streptavidin liposomes. This approach has certain merits. Both components are stable, and can be purified, for instance, by size exclusion chromatography. Because a small number of unbound peptides would not be harmful to the final product, further purification is not required. Oligonucleotide spacers for attaching peptides also are compatible with streptavidin liposomes.
5. Linkage of peptides to the Delivery vehicles a. PEGylation of Peptides for linkage to lipid particles, such as liposomes
Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine primarily because PEG is a biocompatible, nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhao and Harris, ACS Symposium Series 680: 458-72, 1997). In the area of drug delivery, PEG derivatives are used in covalent attachment (z.e., "PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16: 157-82, 1995). Similarly, PEG has been attached to low molecular weight, relatively hydrophobic drugs to enhance solubility, reduce toxicity and alter biodistribution. Typically, PEGylated drugs are injected as solutions.
Numerous reagents for PEGylation have been described in the art. Such reagents include, but are not limited to, N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3 -hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotri azole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG? butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG- NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardini el al., Bioconjugate Chem. 6:62-69, 1995; Veronese et aL, J. Bioactive Compatible Polymers 12: 197-207, 1997; U.S. 5,672,662; U.S. 5,932,462; U.S. 6,495,659; U.S. 6,737,505; U.S. 4,002,531; U.S. 4,179,337; U.S. 5,122,614; U.S. 5,324, 844; U.S. 5,446,090; U.S. 5,612,460; U.S. 5,643,575; U.S. 5,766,581; U.S. 5,795, 569; U.S. 5,808,096; U.S. 5,900,461; U.S. 5,919,455; U.S. 5,985,263; U.S. 5,990, 237; U.S. 6,113,906; U.S. 6,214,966; U.S. 6,258,351; U.S. 6,340,742; U.S. 6,413,507; U.S. 6,420,339; U.S. 6,437,025; U.S. 6,448,369; U.S. 6,461,802; U.S. 6,828,401; U.S. 6,858,736; U.S. 2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637; US 2004/0235734; W00500360; U.S. 2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925).
In one example, the polyethylene glycol has a molecular weight ranging from about 3 kD to about 50 kD, and typically from about 5 kD to about 30 kD. Covalent attachment of the PEG to the drug (known as "PEGylation") can be accomplished by known chemical synthesis techniques. For example, the PEGylation of protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions.
While numerous reactions have been described for PEGylation, those that are most generally applicable confer directionality, use mild reaction conditions, and do not necessitate extensive downstream processing to remove toxic catalysts or biproducts. For instance, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thus its use limits some of the heterogeneity of the resulting PEG- protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite to the terminal methoxy group is generally necessary to accomplish efficient protein PEGylation, with the aim being to make the derivatized PEG more susceptible to nucleophilic attack. The attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines also can react (e.g., the N-terminal alpha-amine or the ring amines of histidine) if local conditions are favorable. A more directed attachment is possible in proteins containing a single lysine or cysteine. The latter residue can be targeted by PEG-maleimide for thiol-specific modification. Alternatively, PEG hydrazide can be reacted with a periodate peptide and reduced in the presence of NaCNBEE. PEGylated CMP sugars can be reacted with a peptide in the presence of appropriate glycosyl-transferases. One such technique is the “PEGylation” technique where a number of polymeric molecules are coupled to the peptide. When using this technique, the immune system has difficulties in recognizing the epitopes on the peptide's surface responsible for the formation of antibodies, thereby reducing the immune response. For peptides introduced directly into the circulatory system of the human body to give a particular physiological effect (i.e., pharmaceuticals) the typical potential immune response is an IgG and/or IgM response, while peptides which are inhaled through the respiratory system (i.e., industrial peptide) potentially can cause an IgE response (i.e., allergic response). The polymeric molecule(s) can shield epitope) on the surface of the peptide responsible for the immune response leading to antibody formation. The heavier the conjugate is, the more reduced immune response is obtained.
PEG moieties are conjugated, via covalent attachment, to the peptides. Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g., Roberts et al., Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see e.g., Guiotto et al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (see e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv. Rev., 54:487-504, 2002). Methods and techniques described in the art can produce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a single protein molecule (see e.g., U.S. Pub. No. 2006/0104968).
As an exemplary illustration of the PEGylation, PEG aldehydes, succinimides and carbonates have each been applied to conjugate PEG moieties, Succinimidyl PEGs (as above) comprising either linear or branched PEGs can be conjugated peptides. PEGylated peptides have been generated using NHS chemistries, as well as carbonates, and aldehydes, using each of the following reagents: mPEG2-NHS-40K branched, mPEG-NHS-lOK branched, mPEG-NHS-20K branched, mPEG2-NHS- 60K branched; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB- 20K, mPEG-SMB-30K; mPEG-butyraldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin. PEGylated peptides have been prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including peptides PEGylated with Dowpharma's p-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30 kDa). Schemes for effecting PEGylation are well known to those of skill in the art. b. Synthesis of Pegylated peptides for conjugation to liposomes
A drawback with many peptide drugs is a short half-life, because they are degraded by proteases that are in the blood. To increase half-life, peptides are chemically coupled to polyethylene glycol (PEG). For purposes herein, the polypeptides can be Pegylated to provide a group for conjugation to a liposome. One end of the PEG moiety is linked to the peptide, and the other end includes a group for conjugation to the liposome. One or more different peptides can be conjugated with one liposome. Typically, PEG is conjugated with an amino group. Amino group can be terminal or in a lysine residue. If the amino group occurs in the active site of the peptide or in a site such that conjugation alters activity, conjugation can reduce or inhibit peptide activity. It is important to select the site for PEGylation so that activity of the peptide is not altered. Detailed herein is a method to achieve this, such that polypeptide is synthesized with PEGylated lysine(s) at the site(s) desired for PEGylation, and any other lysines have conventional protective groups.
In the method herein, the peptide is synthesized in PEGylated form. The lysines are PEGylated, and employed during synthesis of the polypeptide, such as by solid phase synthesis so that a PEG-lysine is incorporated in the peptide chain at a selected lysine residue. Figure 5A schematically depicts a short segment of a polypeptide. Amino acids k, 1, and m are lysines, and that can be separated from each other by several amino acids. In accord with normal peptide synthesis schemes, the terminal carboxylic group is protected by a methyl group that is removed by basic hydrolysis before the next step. The methyl ester of next amino acid is added together with a condensing agent that often is dicyclohexyl carbodiimide (DCCI). The polypeptide often is synthesized on a solid phase surface onto which the growing peptide chain is chemically attached. This allows the washing of the unused reagents and soluble reaction products off. Figure 5B shows a product, in which lysine, residue 1, is PEGylated, and the PEG has an azide group at the end. Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry). If all lysines in a polypeptide occur in the active site or other site for which pegylation affects activity or binding of the peptide, PEG-lysines can be added either in amino or carboxylic end of the polypeptide. This kind of addition can be done to make the binding of the polypeptide with liposome stronger.
When lysine is added into a polypeptide chain, the epsilon amino group must be protected. Protective groups include allyloxycarbonyl and trityl groups. These are orthogonal to other protective groups. For purposes herein in which selected lysines are to be Pegylated for conjugation to a liposome, the epsilon-amino group of lysine is reacted with carboxyl terminated PEG. The bonding is effected via an amide bond that renders the amino group totally inert. As a result, a peptide that contains a PEG moiety in a desired site is produced. The other end of PEG can have a functional group that can be used for the conjugation with a liposome. For example, the functional group can be acetylene that can be reacted with azide that is in the liposome (click chemistry). Depending upon the number of PEG moi eties to be included, other lysines can have conventional protective groups. Thus, no separate PEGylation step is needed; the peptide is synthesized in PEGylated form. Figure 5B depicts a product, in which lysine 1 is PEGylated, and the PEG has an azide group at the end. Azide can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry). Figure 5C shows a PEGylated lysine. c. Liposome preparation for linking the peptides
Figures 2A and 2B depicts a liposome 201 (only the outer atomic layer is schematically shown). The liposome contains 1 - 5 % phosphatidyl ethanolamine that has two fatty acids containing at least 16 carbon atoms, generally 18 - 24 carbon atoms each. A PEG spacer 202 having 10 - 200 ethylene glycol moi eties can bound to each amino group.
These liposomes have one biotin moiety chemically bound per at least 20, 50, 100, or 120, generally about 100 phospholipids. A calculated amount of streptavidin 203 is added. Streptavidin can bind four biotins very strongly; only one or two of these binding sites should be used to bind streptavidin with a liposome, so that two or three binding sites can be used to bind the peptides 205 - 207 (Fig. 2B).
The PEG can contain an N-hydroxy succinimide active ester on one end (Fig. 3), and biotin moiety on the other end. Reagents are chosen so that they are water soluble, somewhat stable in water, and react at room temperature.
Peptides have also amino groups, and PEG spacers can be connected to these amino groups, such as one PEG spacer 204 having a biotin moiety on the other end. Additional PEGs 208 can be added subsequently to provide stealth property against the body’s immune system that tends to remove foreign particles.
Streptavidin coated liposomes provide certain advantages. For example, once liposomes and PEG-peptides are prepared, any and all combinations can be fabricated by mixing the components. The skilled person, such as the physician, can personalize the treatment for the needs and particulars of the treated subject. Personalized regimens of combinations of the therapeutics can be designed and implemented.
Figure 4 depicts another exemplary coupling chemistry in which X is Br or I.
Carbonyl activates the nearby halogen so that it is easily substituted by a strong nucleophile such as a negative sulfur atom. In this case the PEG has a terminal thiol group, and the other end has biotin. The liposome has 1 - 5 % bromo-, or iodoacetyl cardiolipin.
Cardiolipin (For example, see structure below) is an exemplary phospholipid for anchoring the peptides to liposomes and other lipid-based delivery vehicles. Although cardiolipin has been used in liposomes for drug delivery, it generally is not used for anchoring purposes. Cardiolipin liposomes tend to go to the heart, because the heart has more cardiolipin than other organs. To avoid this, a small amount of cardiolipin that is chemically modified is used. Cardiolipin has one free hydroxyl group that will be is esterified with bromo-, or iodoacetyl moiety for the binding of peptides via PEG.
Thiol-PEG is added under slightly basic conditions to form a thioether bond. Streptavidin can be bound as described above. If the PEG has a carboxyl group instead of biotin on the other end, EDC can be used to couple peptides directly with PEG. Cardiolipin has four fatty acids instead of two like all the other phospholipids. This provides stronger anchoring of the peptides to the liposome.
Exemplary of a delivery vehicle, produced as described above and in the Examples, are liposomes displaying a plurality of different peptides including at least two proglucagon peptides and an anabolic peptide. For example, provided are liposomes that display:
PeptideFLl=GLP-l;
PeptideFL4= Oxyntomodulin;
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Peptide); and PeptideMEl=Sermorelin
D. FORMULATIONS AND ROUTES AND MODES OF DELIVERY
The delivery vehicles with linked peptides can be formulated for any suitable route of delivery, including, but not limited to, injection, inhalation, mucosal, and other routes. The route includes the indication treated or tissue targeted and the particular delivery vehicle. Many delivery vehicles cannot be orally administered; some such as certain exosomes can be orally administered. In general, the contemplated route of administration is injection. Pens and syringes containing formulations of the delivery vehicles, as a single dose or multiple doses, are provided. As described herein, the delivery vehicles, such as liposomes, can display a plurality of peptides, such as two or three fat loss peptides and one muscle enhancing peptide, or they can display fewer and mixtures of the liposomes with different peptides can be administered. They can be provided with single peptides linked thereto, and the physician can select combinations of peptides according to a rotational or combinatorial or both regimen, such as any described herein.
E. COMBINATORIAL THERAPY AND ROTATIONAL COMBINATORIAL THERAPY
It is understood that for purposes herein, the peptides are provided on delivery vehicles, such as liposomes. For weight loss and also for diabetes, the delivery vehicles can display at least two peptides for weight loss, and at least one for muscle enhancement, or combinations of delivery vehicles each displaying one peptide are administered together. The regimens as described below administer that therapeutics displayed on or linked to or embedded in delivery vehicles.
Individuals treated with therapeutic agents for conditions, disorders, or diseases, such as chronic conditions, disorders, and diseases, are at risk of developing a tolerance or resistance to the therapeutic effects of the medications. In the context of biological systems and pharmacology, the activation of cell surface receptors can trigger regulatory processes that restrict signaling duration and/or strength. Downregulation is a process by which a particular cellular component, such as a protein (e.g., receptor), decreases in abundance or decreases activity responsive to an external stimulus (z.e., long term administration of an agonist). For example, the expression of a specific receptor or decrease in total receptor number in the cell can decrease (z.e., by enhanced receptor degradation or decreased receptor synthesis or ligand-induced internalization of receptors (endocytic downregulation)). In response to increased or long-term exposure to an agonist, such as a hormone or neurotransmitter or other signaling molecule, the decrease in receptor activation or number can reduce cell sensitivity to the agonist. In the presence of sustained ligand stimuli, the receptor system enters a refractory state thereby preventing the cell from over-responding to the ligand.
This negative feedback mechanism is important in drug treatment, where chronic exposure to a drug and/or therapeutic molecule can become less effective upon long-term or chronic exposure. An example of this negative feedback mechanism are hormone receptors feedback systems. When receptors have been chronically exposed to an excessive amount of a ligand or exposed to a ligand for a prolonged period of time, either from endogenous mediators or from exogenous drugs, this can result in ligand-induced desensitization or internalization of that receptor. Downregulation, therefore, effectively minimizes overstimulation of a pathway to prevent disruption of an organism’s internal processes as a result of the increased or chronic activation.
Downregulation is a cellular mechanism characteristic of long-term administration of therapeutic agents. Understanding downregulation and cellular responses to chronic drug exposure is fundamental to developing an effective drug regimen for treating a complex disease state. Targeted therapeutics that are rotated to decrease receptor or cellular downregulation, or other cellular responses that decrease efficacy or activity of a therapeutic agent can be used to treat diseases, disorders, and conditions, such as obesity, and have wide reaching implications across medical specialties for drug development for chronic conditions in which there are a plurality of targets for therapeutic intervention for in which desensitization to treatments occurs.
Combinatorial treatments involve the use of two or more, generally at least three, different treatments that target different pathways for treating a disease, disorder, or condition. The combinatorial treatments, particularly if they start to lose effectiveness for treatment of chronic diseases, disorders, and conditions, can be rotated, which avoids the problems, particularly desensitization, which occurs with long-term therapies for diseases, disorders, and conditions, such as obesity, Parkinson’s disease, and other chronic diseases, disorders, and conditions that require treatment for many months, and generally for life.
Rotational combinatorial therapy involves protocols/regimens for treatment of a disease, disorder, and/or condition, in which two or more different therapies (or therapeutics) in combination are administered such that the combinations of the -n- therapies are rotated for predetermined or multiple rounds of treatment. Rotational combinatorial therapy or pharmacology is a protocol for treating a disease, disorder, and/or condition, in which a number of therapeutics or treatments, at least two, different therapeutics/treatments for a disease are administered according to a schedule in which different therapeutics and combinations thereof are administered. Each therapeutic/treatment can be different, having a different target or mode of action. There also can be overlap of the therapeutics in the combinations, especially if there is a time period (gap) between therapies (z.e., giving the pathway time to ‘recover’ or regain the ability to become activated). As set forth below, a combination in the rotational protocol can contain more than one therapeutic, wherein each targets the same pathway to effect a therapeutic effect, which can, in some instances, achieve a synergistic effect such that lower doses, compared to monotherapy, of each therapeutic can be administered. In these examples the timing for rotation can be decreased. Because the medications are used in a therapeutic combination, in some examples a medication or medications in the combination can be used at lower dosages than treatment with a monotherapy, to reduce the risk or severity of adverse side effects.
In accord with rotational combinatorial protocols, each combination of treatments is administered for a predetermined time, generally at least a week, and then replaced by a different combination, which is administered for a predetermined period of time. Each combination can include a drug in common, and/or one of the rotations can include only a single therapeutic, but each combination is different from the other combinations. A rotational protocol can include at least two different combinations that are rotated, and generally includes at least three combinations of therapeutics and treatments that are rotated. The protocol can be repeated a plurality of times.
The predetermined rounds for treatment and timing for treatment can be modified if the subject demonstrates signs or signals that a pathway is downregulated, or the subject becomes less responsive to the therapeutic effects of the therapy or the subject exhibits an increase in adverse side effects. For example, the combinations can be switched prior to the predetermined time. In other examples, if the subject exhibits a decrease in the side effects of the medication, this can indicate that downregulation of the pathway has occurred or is occurring, and the dosage can be increased or another therapeutic that activates a different pathway can be substituted. For example, if administration of a medication results in a side effect of jitteriness, and after a set period (z.e., 2 months) the jitteriness diminishes, the diminution can indicate that the therapeutic pathway is downregulated and medication that acts through a different pathway can be substituted.
The regimen can involve a first round in which one, two, or more of the therapeutics/treatments are administered for a period of time, followed by second round in which a different therapeutic/treatment or different combination of therapeutics is/are administered for another period of time, and repeating round one and round two, or administering a third round of pharmaceuticals for a predetermined period of time.
Depending on the number of therapeutics that are part of the treatment, as well as the disease, disorder, or condition, and particulars of the treated subject, different combinations of therapeutics are administered for each time period, providing different combinations of therapeutics rotated for predetermined periods of time. In some examples, the time periods for treatment are not predetermined; the different combinations are administered for each period of time, and the combinations of therapeutics are rotated when the patient is showing reduced therapeutic benefit(s), or the patient is showing increased or intolerable side effects from the therapeutics. The therapy involves at least two different therapeutics, administered sequentially, and then together, or administered together, and typically involves at least three different therapeutics, usually drugs, administered separately or in combinations. Generally, all combinations and orders of administration can be included in the protocol. Hence, the name rotational combinatorial pharmacology.
A regimen of a rotational combinatorial therapy described herein comprises two or more different therapeutic combinations (also referred to as clusters), where each combination includes two or more medications or treatments. Each combination is rotated for a predetermined time of treatment. Rotation can revert to combination one or any other combination in the regimen.
In some examples, a rotational combinatorial treatment or therapy includes the administration of a first therapeutic agent or combination of therapeutic agents and, after a set time point and in fixed intervals, administration of a different therapeutic agent or combination (cluster) of therapeutic agents, where the second combination (cluster) of agent(s) target a receptor/and or pathway distinct from at least one of the initial therapeutic agent(s), and, optionally, any subsequent combinations are administered after a set time point and in fixed intervals. In exemplary regimens described herein, the first therapeutic agent(s) combination is/are administered and then, such as three months later, a second therapeutic agent(s) combination is/are administered. In examples, there is a third agent(s) combination administered after the second agent(s), at a set time point and after the fixed interval. In some examples, there are multiple rounds of administration of agent(s) at fixed time points after the fixed interval(s). Treatment can proceed for years, and can, if necessary be administered for life. Any of the therapeutic agents used in the rotational therapy herein can be a pharmaceutical or non-pharmaceutical therapy.
In some examples, the combinations can be administered in accord with any of the regimens set forth in the any of following tables:
Table 1. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals
Table 2. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals
Table 3. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at One Month Intervals
Tables 4 and 5, below, set forth rotational combinatorial therapy regimens that are rotated at 4-month and 6-month intervals, respectively. In some examples herein, downregulation generally occurs after at least 3 months and, thus, rotations of combinations generally occur at or about 3 months or later. In some examples, such as where downregulation of particular pathways is slow or takes longer than 3 months, regimens such as those set forth in Tables 4 and 5 are employed.
Table 4. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at Four Month Intervals
Table 5. Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at Six Month Intervals
Regimens for rotating and administering therapeutic combinations can be developed in accord with the description provided herein, and by modifying regimens described herein and those set forth in the following tables:
Table 6.1: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals
Table 6.2: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals
Table 6.3: Exemplary Regimen for a Rotational Combinatorial Therapy Rotated at three-month intervals
Exemplary of therapeutics in Tables 6 are the following:
Therapeutic #1=GLP-1
Therapeutic #2=Adiponectin
Therapeutic #3=Leptin Therapeutic #4= Oxyntomodulin
Therapeutic #5=Sermorelin
Therapeutic #6= Peptide YY (PYY)
Therapeutic #7=Amylin
Therapeutic #8=tesamorelin Therapeutic #9=Pancreatic polypeptide (PP)
Therapeutic #10=Enterostatin/GIP
Therapeutic # 11 = Cholecystokinin (CCK)
Therapeutic # 12= Vasoactive Intestinal Therapeutic
Therapeutic # 13=Glicentin Table 6.4
The tables above and description herein provide exemplary of regimens for administration of therapeutic combinations, and rotational combinations for weight loss as exemplary of a condition that can be treated by combinatorial and rotational combinatorial methods and regimens provided herein. The combinations, therapeutics in the combinations, dosages, timing and other aspects of the regimens can be modified as required to achieve a therapeutic benefit and/or decrease adverse side effects. The skilled person can select from among medications that have the recited function and combine them to achieve additive and synergistic effects.
The combinatorial treatment or therapy and rotational combinatorial treatment or therapy provides an improved treatment of diseases and disorders compared to treatment with monotherapies, such as disease states that result from derangements of multiple molecular or cellular pathways or a that show a decrease or plateau in the efficacy of the monotherapy; the combinatorial therapy and/or rotational combinatorial therapy described herein modulates the multiple pathways derangements to ameliorate negative effects of the disease or disorder. Modulation of multiple pathways provides improved therapeutic benefits compared to monotherapy or even dual therapy, which target fewer molecular or cellular targets or pathways. Rotating multiple therapeutics (z.e., pharmaceuticals) is an effective method of preventing molecular and cellular adaptation that occurs after continued (z.e., longterm) treatment with monotherapy. Rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with combination therapy (z.e., treatment with more than one therapeutic) that is not rotated. For example, rotating multiple therapeutics or therapies also can prevent cellular adaptation that occurs after therapy with a monotherapy or combination therapy that is administered for a prolonged period (e.g., longer than 3 months).
In some examples the therapeutics in the combination therapy and/or rotational combinatorial therapy are synergistic; one therapeutic (z.e., drug) enhances the clinical activity of another therapeutic (z.e., drug) when used in the combination, and the cumulative effects of the therapeutics exceed the expected clinical benefit of the sum of the multiple drugs in the combination. In other examples, increased efficacy of the combination works through independent drug action rather than a synergistic effect of the therapeutics in combination; the therapeutic benefit is attributable to a single therapeutic in the combination and the benefits over monotherapy are due to increasing the odds that the combination includes a drug that is effective for a particular patient.
Provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions, such as conditions lasting more than three months, and conditions where patients develop a tolerance to treatment. The rotational combinatorial therapy is for treating chronic conditions with a plurality of known treatments or known pathways associated with disease progression or pathology. A rotational combinatorial therapy regimen can be developed by identifying known treatments/therapies the disease(s), disorder(s), and/or condition(s) for treatment; identifying the pathways, mechanism of actions or targets for treatment; selecting treatment(s) and/or therapies that include treatment(s) and/or therapies that activate different pathways or that have different mechanisms of action; creating combinations that include at least two therapeutics known to activate different molecular and/or cellular pathways and design a regimen for administration of the combinations; and creating a regimen to administer multiple rounds of treatment, with different combinations. In some embodiments, combinations used in one or more rounds of rotation can include a single therapeutic. Generally, all combinations that are rotated include two or more therapeutics or treatments.
1. Identification of diseases, disorders, or conditions for treatment
The combinatorial rotational therapy provided herein can be used in methods of therapy for treating diseases, disorders and/or conditions where the affected patients are at risk of developing a tolerance or resistance to the therapeutic effects of the therapeutic(s) (z.e., medications). The diseases, disorders, and/or conditions have more than one target/pathway for therapeutic intervention. Generally, the disease, disorder, or condition is chronic or requires extended or life-long treatment. Other diseases, disorders and/or conditions are those where the afflicted individuals are at risk of developing a tolerance or resistance to the therapeutic effects of the medications and include chronic conditions, where the disorder or its effects are persistent or long-lasting, or is a disease that develops over time. Patients with a variety of diseases or conditions (e.g., chronic conditions) can benefit from a rotational combinatorial therapy described herein. These conditions include, for example those that can be treated by: 1) a combination of more than one medication and 2) a rotational aspect, such as conditions where the prescribed therapeutics have the potential to decrease efficacy over time. Thus, the methods herein, address the problems of inadequate effectiveness of a monotherapy, and the desensitization. Combinations of a plurality of drugs can improve effectiveness; combining this with rotation of the combinations of the drugs (or of the drugs), leads to sustained weight loss.
A disease, disorder, or condition for treatment with a rotational combinatorial therapy described herein is one where 1) there are a plurality of different therapeutics/treatments known or that can be developed for treating the disease, disorder, or condition, such as where there are a plurality of therapeutic intervention pathways or targets; and/or 2) the disease, disorder, or condition generally is chronic; and/or 3) the disease, disorder, or condition is one for which treatment often fails because tolerance to the therapeutics/treatments develops and/or the therapeutics/treatments become ineffective over time. a. Disease, Disorder, or Condition with a Plurality of Known Treatments
The rotational combinatorial therapy provided herein can be used for treatment of any condition where there is an effective treatment or a plurality of different known therapeutics and/or treatments. For example, the rotational combinatorial therapy provided herein can be used for treatment of any condition where there is a known pharmaceutical or plurality of pharmaceuticals that ameliorate(s) symptoms of the condition. The rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, such that a therapeutic can be developed for treating the disease or disorder or condition For example, a disease, disorder, or condition in which there are a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways that are involved in disease progression can be treated with the rotational combinatorial therapy described herein. For example, the rotational combinatorial therapy described herein can be used to target multiple pathways that are associated with a disease or disorder, but where a pharmacotherapy has not yet been developed.
In disorders involving a host of cellular receptor(s), activation of the receptor(s) can lead to activation of an array of cellular pathways. Administration of a monotherapy, which activates or effects activation of a single pathway can have limited efficacy for improving the disorder. An approach for reducing the symptoms or effects of the disorder can require administering a combination of compounds targeting the different pathways and processes.
The combination therapies described herein can include treatments and/or therapies that target multiple pathways, and diseases or conditions for treatment where targeting a single pathway or mechanism will not confer significant or complete amelioration of the disease state or symptoms. For example, the rotational combinatorial therapy provided herein can be used to diseases where administration of a single therapeutic in animal models or in human patients has a limited therapeutic effect.
In some examples, the disease, disorder, and/or condition is a multifactorial process where the therapeutic benefit from a combination of different therapeutics exceeds the therapeutic benefit of a monotherapy. For example, the disease, disorder, or condition is influenced by multiple genes (polygenic) and resultant genetic pathways, and generally in combination with lifestyle and environmental factors, such as exercise, diet, or pollutant exposures. The combination of genetic and environmental factors act together in concert to trigger the development and progression of the multifactorial disease.
The concept of rotational and/or combinatorial pharmacology provides a method for treating complex disease processes, such as obesity, which involve multiple pathways, in which monotherapy and dual therapy have shown limited success. The combinatorial pharmacology for weight loss as exemplified herein is exemplary of a condition that can be treated with a rotational therapy. Other conditions that involve multiple molecular and/or cellular pathways also can be treated by rotational combinatorial pharmacology, such as conditions where monotherapies are inadequate. Combination therapy also can overcome limitations of monotherapies, where multiple medications, by virtue of the number of medications in the combination, increases the chance the subject is responsive to a particular therapeutic. b. Chronic Conditions
Chronic conditions require ongoing therapeutic intervention and can negatively impact or limit the daily activities or the quality of life of the affected individual. Chronic conditions include conditions in which known therapeutics are ineffective or that do not significantly or completely ameliorate the symptoms or characteristics of the condition. Chronic conditions also include conditions where patients treated with monotherapies are not responsive to treatment or who have not improved on the monotherapy or who continue to have adverse symptoms of the condition despite the therapeutic intervention. Chronic conditions also include conditions where patients treated with combination therapies (z.e., continuous and/or prolonged administration of more than one therapeutic) are not responsive to treatment or who have not improved on the combination therapies or who continue to have adverse symptoms of the condition despite the therapeutic intervention.
Chronic conditions are conditions that persist over time. In some examples, chronic conditions last more than 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more. The rotational combinatorial therapy provided herein is for treating chronic conditions. In some examples, the rotational combinatorial therapy described herein is for treating a chronic condition, disease, or disorder in which the course of the condition, disease or disorder lasts more than about three months. A chronic condition also can be characterized by the amount of time the therapeutics for treating the condition are administered. For example, chronic conditions can be conditions in which therapeutic(s) for treating the condition is/are administered for an extended period of time (ie., about 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more).
Patients with chronic conditions can be administered a medication or combination of medications for months or years, during which time they can develop a decreased response to the medication(s), such as decreased response due to signal downregulation or desensitization to the medication, and can suffer from side effects from prolonged use of the medication(s). Subjects with chronic conditions can benefit from treatment with combinations of therapeutics that are rotated, which can increase the likelihood of a prolonged therapeutic response compared to treatment with a monotherapy or combination of therapeutics that are not rotated.
Provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions. In some examples of chronic conditions, patients are administered therapeutic treatment(s) for extended periods of time and can develop a tolerance to treatments, such that the therapeutics for treating the conditions become or are less effective. For example, provided herein are combination therapies for use in a rotational regimen for use in the treatment of chronic conditions, for use in the treatment of overweight or obesity, and/or for weight loss. The combination therapies for use in a rotational regimen also are for use for treating or ameliorating the symptoms of comorbidities of overweight and/or obesity. Exemplary of such comorbidities include diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, depression, non-alcoholic fatty liver disease, and cancer, such as but not limited to, pancreatic cancer, breast cancer, prostate cancer, gastric cancer, colon cancer, ovarian cancer, head and neck cancer and others. The combination therapy and rotational regime can be modified to decrease the amount or severity of adverse side effects associated with administration of the therapeutic(s). For example, the dosage of the therapeutic(s) can be lowered, or the therapeutics or therapeutic combinations can be rotated with greater frequency.
Rotating combinations of therapeutics can expose patients to each of the particular therapeutics for a shorter amount of time or to a lower dosage, which can each decrease the chance of developing side effects or the severity of side effects from the particular medication. The rotational combinatorial therapy provided herein can be used to minimize side effects during treatment or therapy for diseases or disorders, compared to treatment with a monotherapy or dual therapy or combination therapy with two or more therapeutics. In some examples, the combination therapy described herein can be used and/or administered at a lower dosage than the dosage of the therapeutic used for monotherapy. For example, the combination therapy is used at a lower dosage than the monotherapy which results in fewer or less severe adverse side effects during treatment.
Patients with a chronic condition who previously were treated with a monotherapy, where the monotherapy did not decrease disease signs or symptoms or where the monotherapy did not inhibit or stop disease progression, can be administered a combination therapy or rotational combinatorial therapy provided herein to improve the therapeutic response or to decrease the adverse symptoms associated with the disease state. In other examples, patients with a chronic condition who were previously treated with a combination therapy, where the combination therapy did not decrease disease signs or symptoms or where the combination therapy did not inhibit or stop disease progression, can be administered a rotational combinatorial therapy provided herein.
A rotational combinatorial therapy also can be used for treating subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy and who are responsive to the treatment, but where the treatment did not completely ameliorate symptoms of the condition or did not cure the disease. In these examples, a patient can benefit from a combination therapy targeting multiple molecular and/or cellular to target pathways to which the subject is more responsive. A combination of therapeutics administered in a rotation or regimen described herein can show improved amelioration of symptoms of a chronic condition compared to a monotherapy or combination of therapeutics that are not rotated by decreasing downregulation of therapeutic pathways and activating molecular pathways that were not previously activated (z.e., not previously subject to downregulation). c. Conditions where Patients Develop a Tolerance to Treatments
Patients with chronic conditions can develop a decreased response (tolerance) to the medication(s) prescribed for treating the condition or for ameliorating symptoms of the condition. Such decreased therapeutic response over time can be related or due to signal downregulation or desensitization to the medication(s). Patients that develop a tolerance to a monotherapy or combination therapy can benefit from a rotational combinatorial therapy to overcome the desensitization, such as desensitization due to receptor downregulation.
A rotational combinatorial therapy, thus, can be used for treating conditions where patients develop a tolerance to treatments. For example, subjects with chronic conditions who have previously been treated with a monotherapy or a combination therapy who are responsive to the treatment (z.e., showed an amelioration of symptoms) for a limited time period but where the treatment was less effective or the subject failed to respond to the treatment after a time period can be treated with a combinatorial rotational therapy. For example, a subject who initially showed amelioration of symptoms of the disease or condition, but where the symptoms increased after prolonged treatment, such as, for example, a subject who showed amelioration of symptoms but where symptoms increased after at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment.
2. Development of a Combinatorial Rotational Therapy Regimen
Rotational combinatorial therapies provided herein are for treating a disease, disorder, and/or conditions where there are a plurality of different therapeutics/treatments known or that can be developed for treating the disease, disorder, or condition, and the disease, disorder, or condition generally is chronic and/or is one for which treatment fails because tolerance to the therapeutics/treatments develops or the therapeutics/treatments become ineffective over time so that ultimately treatment of the disease, disorder, or condition fails or becomes increasingly ineffective.
In examples, development of a rotational combinatorial therapy can include a protocol including:
1) identify known treatments/therapies for each disease state or pathways associated with each disease state;
2) identify the pathways, mechanism of actions or targets;
3) select treatments/therapies that activate different pathways, have different mechanism of actions or targets, and/or that are compatible with a rotational therapy; 4) create combinations that include at least 2 therapeutics known to activate different molecular and/or cellular pathways and design a regimen for administration of the combinations; and
5) create a regimen to administer multiple rounds of treatment, with combinations of therapeutics.
Each of these aspects is discussed in turn as follows.
1) Identify known treatments/therapies for each disease state or pathways associated with each disease state
A protocol for developing a rotational combination therapy can include identification of treatments or therapies that are known and/or have previously been characterized for treating a disease, disorder, or condition. For example, a protocol for developing a rotational combination therapy described herein can include identifying treatments or therapeutics that have previously demonstrated activity or efficacy for treating the disease, disorder, or condition or for treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition. For example, a therapy or therapeutic is a drug that is approved by the Food and Drug Administration (FDA) for human use in the United States. In other examples, the therapeutic can show efficacy for treating the disease, disorder, or condition or treating or ameliorating secondary effects or symptoms associated with the disease, disorder, or condition in animal models.
In other examples, the therapeutic (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that was previously identified as a therapeutic that alters (z.e., activates or inhibits) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and/or ex vivo model systems. The in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug/pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is/are implicated or use of a particular therapeutic is predicted to be effective. Studies or demonstrations that a therapy or therapies is/are effective for treating or ameliorating a disease, disorder, or condition can be used to select therapeutics for inclusion in the rotational combination therapy described herein. In other examples, the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be identified in screens for effective therapeutics for treating a disease or disorder. For example, in vitro screens in model systems that replicate disease characteristics can be conducted for new substances showing activity and additionally for assessing effectiveness for treating a disorder. In some examples, the therapeutic(s) (z.e., pharmaceutical) for inclusion in the rotational combination therapy described herein are identified in vitro in screens conducted in cell culture, in a Boyden chamber, in three-dimensional cultures, in microfluidic systems, using 3D bioprinting, or in other systems that can be used to identify therapeutics that can be for treating a disease, disorder, or condition with the rotational combinatorial therapy described herein.
In examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, known weight loss medications, such as weight loss medications described herein can be included in the combination. In some examples, a previously identified weight loss medication can be included, including one or more of mitochondrial uncouplers, amphetamines, thyroid hormones, drug cocktails, neuromodulators, lipase inhibitors, cannabinoid receptor antagonists, gastrointestinal-derived peptides chemically optimized for pharmaceutical use, and others. In some examples, one or more FDA approved medications can be included in the combination therapy and/or incorporated into a liposome, generally into a liposome that displays one or more of the peptides. For example, one or more of orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, tirzepatide (is a GIP analog that acts on GIP and GLP-1 receptors; sold under the trademark Mounjaro®) dual agonist, and semaglutide can be included in the combinations described herein for treating obesity or overweight or for effecting weight loss. In other examples, FDA approved medications that are for use for a disease or disorder where the subjects exhibit weight loss as a result of the therapeutic regimen, but for which weight loss is not the primary objective, can be included in the combination therapy provided herein, for effecting weight loss.
2) Identify the pathways, mechanism of actions or targets
A protocol for developing a rotational combinatorial therapy can also include identification of pathways, such as, for example, molecular or cellular pathways, associated with the disease, disorder, or condition. The molecular and/or cellular pathways can be used to determine the therapeutic(s) for inclusion in the combinatorial therapy.
Treatments or therapeutics for inclusion in the rotational combinatorial therapy described herein can be selected from compounds that target a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition. Treatments or therapeutics can activate a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that activation of the pathway ameliorates symptoms of the disease, disorder, or condition or secondary side effects. Alternately, treatments or therapeutics can inhibit a pathway associated with the disease, disorder, or condition; or symptoms, or secondary or side effects of symptoms associated with the disease, disorder, or condition, such that inhibition of the pathway (z.e., inhibition of an overactive pathway or an off-target pathway) ameliorates symptoms of the disease, disorder, or condition or secondary side effects.
When identifying a pathway for targeting in the rotational combinatorial therapy described herein, biomarkers can signal an abnormal process or a condition of the disease. Biomarkers can be used to identify targets for treatment and potential responses to therapeutics and therapeutic combinations. For example, biomarkers can be used to predict a patient’s response to individual therapeutics (z.e., drugs) in a combination. For example, higher receptor expression in a patient can correspond to a better response to a corresponding therapy, and lower expression of receptor activators can indicate a higher or lower chance of responsiveness to a particular therapeutic(s) (z.e., drugs). Thus, biomarker identification and characterization, and use of validated biomarkers (z.e., biomarkers of a particular disease state or status) can help predict patient response and therapy -related side effects and inform selection of therapeutics for inclusion in the combination and/or rotational combinatorial therapy herein.
The skilled artisan can evaluate the literature, for example articles published in the scientific literature, such as, for example, in the US National Library of Medicine and the National Center for Biotechnology Information, which assembles biomedical literature from MEDLINE, life science journals, and online books. The scientific literature can identify and assess molecular and cellular pathways for treating diseases, disorders, and/or conditions described herein. Molecular targets for treating a disease, disorder, or condition can be identified and/or evaluated in the scientific literature and used to determine molecular targets for therapeutics for treating the conditions identified and described herein.
In other examples, a pathway associated with the disease, disorder, or condition or symptoms thereof for treatment with a combination or rotational combinatorial therapy described herein can be identified using in vitro, in vivo, and/or ex vivo model systems. The in vitro results or results from model systems can indicate that targeting a particular pathway or pathways or use of a particular therapeutic (i.e., drug/pharmaceutical) or treatment will be effective for treating subjects (i.e., human patients) in which the pathway or pathways is implicated or use of a particular therapeutic is predicted to be effective. Studies or demonstrations that a molecular and/or cellular pathway plays a role in pathogenesis or progression of a disease or condition can be used to determine therapeutics for including in the combination and rotational combination therapy described herein. The therapeutic (i.e., pharmaceutical) for inclusion in the rotational combination therapy described herein can be one that alters (i.e., activate or inhibit) a pathway associated with the disease, disorder, or condition or symptoms thereof in vitro, or in in vivo and/or ex vivo model systems.
In examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, molecular and/or cellular pathways that are altered in gastric bypass patients can be targeted in the therapy by combining therapeutics that target each pathway. For example, molecular and/or cellular pathways that are altered following gastric bypass or other surgical weight loss procedures can be used to formulate a pharmacotherapy for inclusion in the combination and/or rotational combination therapy described herein. For example, neuro-hormonal gut peptides that are altered following bariatric surgery; or exogenous (i.e. rationally designed) peptides that mimic gut peptides or that are gut peptide receptor agonists; or therapeutics that alter expression and/or activation and/or activity of neuro-hormonal gut peptides that are altered following bariatric surgery, such as, for example, therapeutics that increase expression and/or activation and/or activity of neuro-hormonal gut peptides that are increased following bariatric surgery, can be included in the combination therapy described herein. The combinatorial regimens and the rotational combinatorial regimens can mimic the effects of bariatric surgery, particularly gastric bypass surgery, such as the Roux-en-Y surgery. Gastric bypass surgery has effects that include decreased absorption of food, including intestinal absorption, on glucose homeostasis, and results in hormonal changes that alter appetite/satiety and energy consumption and other physiological processes. Rerouting food through the gastrointestinal tract leads to changes in gut hormone secretion. Changes in gut hormone levels after RYGB, include increased anorectic hormones, such as GLP-1 and PYY, which induce satiety, and decreased levels of orexigens, such as ghrelin, an appetite-stimulating hormone. The rotational combinatorial methods herein allow a variety of combinations of drugs/treatment to reduce desensitization, and adverse effects, while providing the advantages of gastric bypass surgery by combining administration of various drugs that reduce appetite, and hormones that alter appetite. Combinations of such drugs and treatments are detailed herein and are exemplified in the working examples and throughout the description.
Provided are combinatorial methods and combinatorial rotational methods in which combinations of medications, such as peptide hormones or agonists thereof and/or antagonists of peptide hormones that are reduced following bariatric surgery, are administered. Particular combinations are described herein and exemplified in the working examples.
In other examples of a rotational combinatorial therapy for treating obesity or overweight or for effecting weight loss, molecular and/or cellular pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and or therapeutics for the combination therapy.
3) Select treatments/therapies that activate different pathways, have different mechanism of actions or targets, and/or that are compatible with a rotational therapy
Combination therapies containing at least two, and generally at least three, combinations of a plurality of therapeutics and rotated to prevent desensitization of activated pathways exhibit efficacy that is greater than a monotherapy or combination therapy that is not rotated. The combinations contain therapeutics that activate or are known to activate different molecular or cellular pathways. For example, the combinations contain therapeutics that activate or are known to activate a total of at least two, three, four, five or more different molecular or cellular pathways. By virtue of rotating the therapeutic combinations at set intervals, where the combinations contain therapeutics that target different molecular pathways, there is a decreased probability of downregulation of the pathways and, thus, a decreased chance of lower therapeutic effect.
Therapeutics that decrease in efficacy over time can be included in the combinations herein, including, for example, pharmaceutical medications that activate molecular pathways that are downregulated, leading to decreased efficacy of the pharmaceutical. The combinatorial therapy herein includes rotating from one combination comprising a plurality of therapeutics to a next combination comprising a plurality of therapeutics. The rotational aspect overcomes limitations associated with therapeutics that decrease in efficacy over time; the switch to a new combination results in a continuous therapeutic effect that does not wane, or wanes less than treatment with a monotherapy containing one of the therapeutics in the combination.
In examples, therapeutics for inclusion in the combination for the rotational combinatorial therapy herein are those that do not have severe adverse side effects upon ceasing use of the particular therapeutic. For example, therapeutics that do not induce withdrawal symptoms as a result of ceasing or reducing use of the therapeutic (z.e., medication). When selecting medications for inclusion in the combinations, medications that induce withdrawal syndromes and rebound upon discontinuation can be avoided. Therapeutics for inclusion in the rotational combinatorial therapy are those with a low chance of withdrawal or adverse events upon switching to the next combination of medications or those that do not induce adverse events upon switching to the next combination of medications.
In examples, therapeutics for inclusion in the combinatorial therapy are those that are not delayed in producing a therapeutic effect or are not delayed in ameliorating symptoms of the disease or condition to be treated. The therapeutics included in the rotational combinatorial therapy are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or more, and in some examples rotated at least every 3 months. In these examples, the therapeutic(s) takes effect (z.e., has activity) or the therapeutic effect(s) is/are produced or amelioration of symptoms begins near to the time of initial administration, such as within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more of treatment. For example, the therapeutic(s) takes effect (ie., has activity) or the therapeutic effect(s) is/are produced or amelioration of symptoms begins within at least 1 week of initial administration. The therapeutic activity can begin within the first half, third, quarter, or tenth of the rotational time period. For example, for a rotational combinatorial therapy where the combinations are rotated at 3-month intervals, the therapeutics in the combinations can show therapeutic activity within 1 month, and more generally within at least 2 weeks or 1 week or less than 1 week, such as two or three days after initial administration.
Medications that are toxic if administered at high dosages can be included in the rotational combinatorial therapy described herein. Therapeutics (ie., drugs) that show toxicity or adverse side effects when administered in therapeutic amounts can be included in the combination therapy described herein; the therapeutics used in combination with other, complementary, therapeutics can be administered in lower dosages such that when they are administered in combination with other therapeutics, and in a rotational regimen as described herein, they effect amelioration of symptoms or treatment of the disease, disorder, or condition without the adverse effects. Administration of therapeutics in a rotational regimen described herein can reduce drug toxicity, and adverse side effects, while maintaining or improving clinical efficacy. In some examples, a particular medication in the combination is administered at a lower dosage than the medication would be administered in a monotherapy; administration of the medication at the lower dosage decreases drug associated toxicity and adverse side effects.
Previous treatment (z.e., with a monotherapy or combination therapy) can be used to predict a patient’s response to a therapeutic or class of therapeutics or to similar therapeutics (ie., therapeutics that act through the same molecular pathway). Previous treatment (z.e., with a monotherapy or combination therapy) also can be used to predict whether targeting a particular molecular pathway will be effective in that patient. Patients who have shown a positive response to therapeutics targeting one molecular pathway, can be treated with a rotational combinatorial therapy containing therapeutics that target that same pathway. Information from patients who were not previously responsive, or did not show amelioration of symptoms, after treatment with a monotherapy targeting one molecular pathway, can be used to determine the therapeutics for inclusion in a combination therapy herein. For example, if a patient was not previously responsive to a particular pharmaceutical, a plurality of pharmaceuticals targeting the molecular pathway can be included in a combination therapy. In other examples, if a patient was not previously responsive to a particular pharmaceutical, the pharmaceutical can be excluded from the combination therapy. In other examples, if a patient was not previously responsive to a particular pharmaceutical, the pharmaceutical can be included in the combination therapy with other therapeutics predicted to act synergistically with the pharmaceutical.
Treatments that target different pathways associated with satiation, metabolism, hunger and weight gain or loss can be included in a rotational combinatorial therapy herein for treating overweight, obesity or for weight loss, or for treating comorbidities of overweight or obesity, such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (z.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, metabolic syndrome, obstructive sleep apnea, cancer, depression, and non-alcoholic fatty liver disease. For example, therapeutics that mimic the physiological effects following gastric bypass can include in a rotational combinatorial regimen as provided herein.
4) Identify combinations that include at least 2 therapeutics known to activate different molecular and/or cellular pathways and design a regimen for administration of the combinations
The combination can include therapeutics that target the same pathways or that target different pathways. Therapeutics that can provide complementary benefits (z.e., modulate similar or the same molecular pathways to compound the effects), or therapeutics that act through different molecular pathways to effect positive benefits through a variety of pathways can each be included in the rotational combinatorial therapy described herein. The therapeutics in the combinations generally target different pathways. Selecting therapeutics that target different pathways is generally to prevent redundancy in the pathways that the therapeutics target, such as, for example, where redundancy does not increase the therapeutic benefit over the benefit of a single therapeutic, or when redundancy can increase the likelihood or severity of adverse side effects. Selecting therapeutics that target different pathways also overcomes the decreased therapeutic effect that occurs after desensitization of a particular molecular pathway after prolonged treatment. For example, if a therapeutic in a first combination targets a pathway that is desensitized after use, the therapeutic in the second combination targets a second pathway, which is not desensitized, and, thus, not downregulated; a third combination can contain the first therapeutic, if the desensitization no longer exists, or can contain a third therapeutic that targets a third pathway. The rotation of therapeutics that target different pathways overcomes the reduced therapeutic effect after desensitization of a pathway.
The therapeutics for use in the rotational combinatorial therapy described herein can be therapeutics that are complementary in that one effects treatment of secondary effects (z.e., inflammation) and the other effects treatment of the primary cause of the disorder. For example, one therapeutic in the combination can inhibit production of proinflammatory cytokines that are a downstream effect of the primary disorder, such as, cancer and obesity. In other examples, the combination can include therapies that treat or ameliorate the symptoms of concomitant disease states, such as a treatment (z.e., medication) for hypertension included in a combination for treatment of the primary condition of obesity.
Therapeutics that can provide complementary benefits by effecting treatment of different disease phases can be selected for inclusion in the rotational combinatorial therapy described herein. For example, in multi-phasic diseases states, one therapeutic or combination of therapeutics can effect treatment or amelioration of effects of an acute phase of the disease and the other therapeutic(s) can effect treatment or amelioration of effects of a chronic phase or state of the disease.
In some combinations that include therapeutics that target the same pathways, the overlap (redundancy) is, for example, to ensure the efficacy of the combination, to ensure activation or inhibition of a particular pathway. Therapeutics with overlapping targets also can be included in separate combinations in the therapeutic regimen, such as, for example, combinations that are administered at different points in the therapeutic regimen, and generally with a cycle of the regimen separating administration of the redundant therapeutics.
5) Create a regimen to administer multiple rounds of treatment, with combinations of therapeutics
The combinatorial rotational therapy described herein includes a regimen for administering the plurality of combinations multiple times, such that there are at least two rounds, and generally at least three, four, five or more rounds of treatment, with different combinations. Various factors can influence the timing for administering the plurality of combinations in the rotational combinatorial therapy provided herein including, but not limited to: the particular therapeutics in the combinations; the particular disease to be treated; the predicted and/or known efficacy of the therapeutics or therapies, alone or in combination with other therapeutics and/or therapies; the time for the therapeutics to demonstrate therapeutic activity (e.g., onset of action); the recommended time for therapeutic administration; the maximum recommended time for therapeutic administration; medication dosages; the characteristics of a patient to be treated, including, but not limited to, gender, weight, age, overall health, comorbidities, and other characteristics; and other particulars of the therapeutic(s), patient and disease, disorder, or condition.
In examples where the rotational combinatorial therapy is for treating a rapidly progressing disease, disorder, or condition, the first combination in the rotational combinatorial therapy can include a therapeutic or therapeutics with a high (z.e., the highest) probability of activity and/or efficacy for treating the disease, disorder, or condition. For treating a disease, disorder, or condition that is less rapidly progressing, where an extended time period for administering a second- or third-line combination therapy can be considered, lower dosages or a less aggressive first combination of therapeutics can be administered. The therapeutics for inclusion in the first line therapy (i.e., therapeutics in the first cycle of the rotation) can depend on the disease, disorder, or condition, to be treated, the disease state, state, the rate of progression of the disease, disorder, or condition, the severity of the disease, disease, disorder, or conditions. As detailed above and below, the skilled physician can modify the therapeutics for inclusion in the rotational combinatorial therapy described herein based on the available therapeutics, and, for example, the disease to be treated, the disease state, the rate of disease progression, the severity of the disease, the particular patient to be treated, and other factors. The rotational aspect of the combination therapy provided herein provides for changing of doses and replacement of ineffective or less effective therapeutics with others that have increase efficacy, for example, based on the patient response.
The time for the drug or therapeutic to exhibit therapeutic activity also can be considered when selecting a drug or other therapeutic for treatment in a rotational treatment regimen provided herein. In one example, a drug or therapeutic used in the rotational combination therapy herein should be active prior to rotation and administration of the next therapeutic combination. For example, if a medication takes one week to show therapeutic activity, the combinations will not be rotated at less than one week. For example, if a medication takes one week to show therapeutic activity, the combinations can be rotated after at least one month, two months, three months or more, in order to show therapeutic activity of the initial combination.
As detailed herein, the timing for rotation of the therapeutic combinations can be determined. Various factors can be considered to identify indicators for a switch from one therapy (z.e., treatment or drug or therapeutic or combination thereof) to another, and the timing for a switch. In some examples, the following factors can influence the decision to switch medications: one or more adverse side effects; change in clinical status (e.g., improvement or worsening of the disorder or condition to be treated); poor drug activity despite administration of a therapeutically effective amount of drug; concomitant pharmacotherapy (i.e., drug-drug interactions); need for a different route of administration (e.g., oral vs. injection), such as, for convenience or for improved adherence to the administration regimen or for drug uptake and/or activity; subject responsiveness to the drug (i.e., poor responsiveness), such as because of receptor down-regulation or due to sex or age or race; and others. Thus, the timing for switching combinations will depend on many factors, including the particular therapeutics in each combination. The information provided herein can be used to develop guidelines for a rotation schedule. In examples, combinations are administered in rotation, for predetermined periods, such as for at least 1 month, 2 months, 3 months or 4 months or 5 months or 6 months or 9 months or 1 year or 15 months or 18 months or 2 years or 27 months or 30 months or 33 months or 4 years or more, and in some examples rotated at least every 3 months. As downregulation can occur after at or about 3 months or less, the combinations can be rotated every three months.
After an administration regimen, such as a rotational regimen described herein, has been implemented in a particular patient, the regimen can be modified to improve the therapeutic effects. The patient’s clinical status can be used to modify the treatment regimen or schedule or drugs administered. For example, if a patient develops drug sensitivities, the regimen can be modified to switch to a new therapeutic under an expedited schedule. In other examples, the rotational schedule can be extended if previously unrealized benefits of the therapeutic are achieved with a particular medication.
As detailed herein, the dosage of the individual therapeutics in the combinations for rotation can be determined. Various factors can be considered to identify the dosage for each therapeutic (ie., drug). The examples and disclosure provided herein can be used to develop guidelines for a rotation schedule, and identify the medications for inclusion in the regimen/schedule. The dose of an individual therapeutic can vary depending on the other therapeutics in the combination, and can vary depending on the doses of the therapeutics in the other combinations in the rotation. The regimen can include guidelines for dose adjustment based on the action of the previously administered medications, relative potency, and other facts that can influence efficacy of the therapeutic(s).
The dose range for each of the individual therapeutics in the combinations provided herein can be adjusted by monitoring the subjects (z.e., patients), such as monitoring plasms or monitoring symptoms or monitoring adverse side effects after administration of the combination(s). The dose administered can be such that the subject will maintain a plasma level to effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered. The dose also can be a dosage that will effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered where the subject does not exhibit adverse side effects, or does not exhibit adverse side effects at a severity that will effect cessation of the treatment.
F. METHODS OF TREATMENT AND USES
The combinatorial and rotational combinatorial therapy provided herein can be used in methods of therapy for treating chronic conditions, such as, for example, overweight and obesity. In other examples, the combinatorial and rotational combinatorial therapy described herein can be used in methods of therapy for treating chronic conditions such as diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (i.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and non-alcoholic fatty liver disease.
In the methods, a combination therapy is administered in a rotational regimen to a subject having a chronic condition. As described herein, the first combination of therapeutics, in a combination regimen with a second (and third or more) combination(s) far exceeds the effects of a monotherapy or combination therapy that is not rotated, for treating a chronic condition, such as, for example, obesity. For example, the effects can be synergistic. For example, extent and level of weight loss observed following the combination therapy provided herein achieves results that have heretofore not been achieved with existing non-surgical weight loss therapies, including increased fat loss and muscle gain that surpasses existing treatment regimens.
1. Therapeutic Uses of the Combinatorial Therapy
Combinatorial and pulsed-dose pharmacology have been used for a variety of diseases, disorders, and conditions, such as pain control, birth control and treatment of cancer, COPD, Parkinson’s disease, Alzheimer’s disease, and bacterial infections. These previous uses do not contemplate administering a combination of at least two, generally, at least three, different therapeutics/treatments (i.e., therapeutics having a different target or different mode of action) according to a schedule in which different therapeutics are rotated, and/or the combinations selected are not selected to target different pathways. Treatment strategies to deal with complex medical issues such as cancer, pain control and pulmonary issues also are described herein to integrate more than one medication (combinatorial pharmacology) administered in a rotational regimen, to improve clinical outcomes. The methods and regimens provided herein are designed to target different pathways involved in disease, disorder, or condition, and to rotate combinations of treatments.
In accord with methods herein, rotational combinatorial therapy for treating complex medical disorders include first identifying known treatments/therapies for each disease state or pathways, and mechanisms of action or targets associated with the disease. Then, treatments/therapies that activate different pathways, have different mechanism of actions or targets, and/or that are compatible with a rotational therapy can be identified. Using previous treatments and pathways/mechanisms/targets associated with the disease state, combinations that include at least 2 therapeutics known to activate different molecular and/or cellular pathways can be selected and a regimen to administer multiple rounds of treatment, with combinations of therapeutics, can be designed. As set forth herein, the methods for selecting therapeutics for inclusion in a rotational combinatorial regimen, and the timing for administration of the therapeutic combinations can be prepared. In examples, the combinations can be administered in rotational regimens for treating, for example, any of the diseases, disorders and/or conditions set forth below. a. Combination Therapies in Cancer
Several cancer therapeutics are used in combination but without a rotational component to treat tumors in patients. For example, ABVD (Adriamycin, Bleomycin Vinblastine Dacarbazine) can be used as the initial chemotherapy treatment for newly diagnosed Hodgkin lymphoma. ABVD has been the most effective and least toxic chemotherapy regimen available for treating early-stage Hodgkin Lymphoma. One cycle of ABVD chemotherapy is typically given over 4 weeks in two doses, where the first dose of the drug combination is administered on day 1 and the second dose is administered on day 15, where all four of the chemotherapy drugs (Adriamycin, Bleomycin Vinblastine Dacarbazine) are administered intravenously.
Another combination therapy for cancer treatment is known by the acronym CHOP, which includes a combination of cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone for the treatment of non-Hodgkin lymphoma. Cyclophosphamide, methotrexate, and fluorouracil (5FU), also known as CMF, also are used in combination for treatment of breast cancer. Other chemotherapy regimens used in the clinical setting include the Stanford V protocol, which generally includes a two to three month treatment period with a chemotherapy regimen (doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin, etoposide, and prednisone) followed by radiation treatment; and the BEACOPP regimen, which includes administering a combination of bleomycin, etoposide, doxorubicin hydrochloride (Adriamycin), cyclophosphamide, vincristine (Oncovin), and procarbazine for treatment of Hodgkin lymphoma. The BEACOPP treatment generally includes four to eight 14- or 21 -day cycles with no drugs given on days 15-21. A course of BEACOPP therapy also can be combined with ABVD. An exemplary BEACOPP therapy protocol is set forth in Table 7, below:
Table 7: BEACOPP regimen
Previous combination therapies for treatment of cancer, including those described above, thus exist. These combination therapies, however, lack a rotational component of rotational combinatorial therapies and regimens provided herein. Any of the previously described combinations of cancer therapeutics can be modified in accord with the rotational combinatorial therapy described herein. For example, to prevent adverse side effects associated with administration of multiple medications on day one (z.e., 5 medications on day one of the BEACOPP protocol), the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications, such as, but not limited to, chemotherapeutics. b. Combination Therapies in Pain Management Some individuals with chronic pain undergoing long- term treatment with painkillers, such as opioids, can develop decreased responsiveness to the opioids despite dose titration (Knotkova et al., J. Pain Symptom Manage. 38(3): 426-39 (2009)). Opioid rotation is a strategy implemented to increase efficacy of the combination of opiates; one opiate is initially administered and when efficacy begins to wane, the first opiate is replaced by another to optimize clinical outcomes and lessen side effects. This drug “exchange” substitutes one opioid for another where both medications work through a similar pathway, both targeting the mu receptor; the medications are “exchanged” rather than rotated.
With pain treatment, opioids are substituted when efficiency begins to wane, not at predetermined intervals, and based on a “shared decision-making approach” between the treating physician and the patient (Fine el al.. J Pain Symptom Manage (2009) 38(3): 418-425). There is no fixed period at which opioids are exchanged, the exchange is based on clinical efficacy which may or may not correlate with the biological process of receptor downregulation.
In contrast, rotational combinatorial therapies described herein exchanges a group of medications for a second group of medications, where the second group targets different pathways or targets than the first group. With opioid rotation, the rotated opioids are the same, and generally is one drug, not a combination of drugs, exchanged for a second drug. Also, in embodiments herein the rotational therapy described herein includes a regularly defined period for the rotation to occur, including regularly defined intervals, to avoid receptor downregulation and to target different receptors. c. Combination Therapies in Oral Contraception
Treatment with oral contraceptives employs both combinatorial and pulse- dosed pharmacology treatment strategies; treatment with oral contraceptives is not rotational, the different medications are not rotated. Oral contraceptives come in a variety of formulations; some formulations contain both estrogen analogs and progestin, and some contain only progestin; some pills are monophasic, delivering the same dose of hormones each day, and others are multiphasic, where the doses vary each day. Doses of the component hormones also varies among commercial oral contraceptive products. The monophasic pills employ a strategy that targets the same, one pathway for birth control each month, with a pulse of hormones, a “pulse-based strategy.”
Multiphasic birth control pills have both and estrogen analogs and progestin components with a fixed increasing dose that is given for set periods of time for a set period (z.e., biphasic dosing includes tablets of one strength for 7 to 10 days, then tablets of a second strength for the next 11 to 14 days; triphasic dosing includes tablets of one strength for 5 to 7 days, then tablets of a second strength for the next 5 to 9 days, and then tablets of a third strength for the next 5 to 10 days). The birth control pills target the same estrogen and progesterone receptors.
Thus, birth control pills can contain a combination of two medications and multiphasic birth control pills can increase in dosage over the month. There is a continuous activation of the same receptors (ie., one or both of estrogen and progesterone receptors) throughout the month, and activation of these same receptors during the next month. Activation of the same set of receptors differs from the rotational combinational therapy described herein because treatment with birth control pills does not contain a rotational element where different combinations of therapeutics (z.e., medications) are administered and rotated to prevent decreased efficacy due to downregulation of receptor activity; each therapeutic in the rotational combinatorial therapy described herein has a different target or mode of action, unlike birth control pills which target the same receptors each month.
Present combination therapies used for treatment of cancer or for pain management or for contraceptive purposes differ from the rotational combinatorial therapy described herein, and can be modified in accord with the rotational combinatorial regimen described herein. For example, efficacy of chemotherapeutic combinations can be improved by applying the rotational combinatorial regimen described herein; chemotherapeutic combinations can be rotated in accord with a specified schedule to target different receptors or pathways to prevent receptor downregulation or decreased efficacy or increased toxicity, such as adverse side effects, which occurs after prolonged continuous use of the single combination. d. Combination Therapies to Treat Pathogens
Regular use of antibiotics, such as treatment with a monotherapy or combination therapy, eventually leads to antibiotic resistance in the targeted microorganism (z.e., bacterium). Combination therapies are strategies for treatment of bacterial infections to overcome or prevent drug resistance, broaden the antimicrobial spectrum, improve the efficacy, treat multi-drug resistant bacteria, and lower the dose of the individual drugs to reduce the side effects. Combination therapy with multiple antibiotics have been described (Drusano et al. (2014) PLos ONE 9 7):el01311; U.S. Patent Publication No. 2021/0236589).
US Patent Publication No. 2021/0236589 describes selecting combinations of three antibiotics and rotating among the three different-member combinations to eliminate the possibility of developing resistance.
The combinations of such antibiotics can be administered in a rotational combinatorial regimen to ameliorate side effects from the particular antibiotics and to avoid development of antibiotic resistance. e. Combination Therapies to Treat Alzheimer’s Disease
There is no cure for the progressive neurodegeneration and resulting phenotypes that occur in patients with Alzheimer’s disease (AD). A wide spectrum of approaches exist for treatment of AD, with the majority focusing on targeting the Ap peptide to slow disease progression (Galimberti el al. , Ther Adv Neurol Disord 4:203- 216 (2011)). Medications and management strategies can improve symptoms, albeit temporarily. Aducanumab (sold under the trade name Aduhelm™) is an intravenous infusion therapy that targets beta-amyloid, and is approved to treat AD. Other medications, such as cholinesterase inhibitors, such as, but not limited to, medications sold under the trademarks Aricept®, Exelon®, and Razadyne®, glutamate regulators, such as, but not limited to the medication sold under the name Namenda®), and combinations of a cholinesterase inhibitor and a glutamate regulator (sold under the trade name Namzaric®). Non-cognitive symptoms, such as behavioral and psychological symptoms, can be treated by orexin receptor agonists (such as the medication sold under the trademark Belsomra®). All of the medications have adverse side effects, including one or more of nausea, vomiting, headache, constipation, confusion and dizziness, impaired alertness and motor coordination, worsening of depression or suicidal thinking, complex sleep behaviors, sleep paralysis, and compromised respiratory function. A combinatorial pharmacological approach has been described to attenuate the microglial activation and chronic inflammation characteristic of AD (McLarnon (2019) Current Alzheimer Research 16: 1007-1017). The anti-inflammatory combination therapy described by McLarnon does not contemplate rotation of the combinations, and includes administration of a cocktail of compounds to modulate inflammatory pathways activated by microglia in response to the proinflammatory AD brain microenvironment. McLarnon does not consider combination therapy for treatment of aspects of AD pathology that are not related to the chronic inflammation, such as pathology that is related to amyloid deposition or other factors, such as amyloid beta activation or altered cell signaling.
Additional medications for inclusion in the rotational combinatorial therapy for treatment of Alzheimer’s are set forth in Table 8, below:
Table 8: Exemplary Medications for Alzheimer’s Treatment
Additionally, these rotational combinatorial treatments for Alzheimer’s disease can include the weight loss regimens or weight loss drugs. For example, glucagon-like peptide 1 (GLP-1) receptor agonists, including those described herein, exhibit neurotrophic and neuroprotective effects in amyloid-P (AP) toxicity models of Alzheimer’s disease (AD). Hence these drugs can be included in a rotational combinatorial regimen for Alzheimer’s disease. f. Combination Therapies to Treat Hypertension
Combination therapies are not generally used to treat hypertension because of concerns about lowering blood pressure to unsafe levels and increased risk of adverse side effects. Generally, if medications are used in combination to treat hypertension, a first medication is administered and a second can be added to the treatment if the first medication is ineffective and/or there are no significant adverse side effects. There are combination therapies have previously been shown to be superior to monotherapy for treatment of hypertension. A combination of losartan at 50-100 mg with hydrochlorothiazide at 12.5 -25 mg lowered systolic blood pressure significantly (p<0.001) more than either drug alone (MacDonald et al.. J of the American Heart Association DOI: 10.1161/JAHA.117.006986 (2017)).
Combinations of medications for the treatment of hypertension generally include two antihypertensive agents with different mechanisms of action that demonstrate enhanced efficacy compared to either medication alone. The medications can be used at lower dosages than treatment with individual medications, to reduce the risk or severity of adverse side effects. Combinations of drugs used for treatment of hypertension are set forth in Table 9, below:
Table 9: Exemplary Medications for Hypertension Treatment
Adapted from Skolnik et al., Am Fam Physician (2000) 61(10):3049-3056
There are combination therapies that have been employed for treatment of hypertension, including those described above, but not rotational of combinations. The medications can be administered initially as a combination of two drugs, or therapy can begin with one medication and one or more medications is added to the monotherapy. Any of the previously described combinations of medications for treatment of hypertension or medications known to treat the molecular and cellular pathways associated with hypertension can be modified in accord with the rotational combinatorial therapy and regimens as described herein. For example, to prevent adverse side effects associated with administration of multiple medications at once or to target alternative molecular pathways, the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications (ie., ACE inhibitors and/or diuretics). g. Combination Therapies to Treat Parkinson’s Disease
Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterized by altered body movements, including tremor, stiffness, slowed movement (bradykinesia), loss of automatic or autonomic movements, and speech changes. PD is not curable; a number of medical treatments are used in the management or improvement of PD symptoms, including levodopa alone or in combination with carbidopa, dopamine agonists, MAO B inhibitors (z.e., selegiline (sold under the trademark Zelapar®), rasagiline (sold under the trademark Azilect®) and safinamide (sold under the trademark Xadago®), Catechol O-methyltransferase (COMT) inhibitors. Entacapone (sold under the trademark Comtan®) and opicapone (sold under the trademark Ongentys®), Anticholinergics, such as, but not limited to benztropine (sold under the trademark Cogentin®), trihexyphenidyl, and amantadine.
Any of the previously described combinations of medications for treatment of PD can be administered in accord with the rotational combinatorial therapy described herein. In other examples, molecular and cellular pathways associated with PD progression and/or symptoms of PD can be targeted with therapeutics for inclusion in the rotational combinatorial therapy herein. For example, therapeutics that target dopaminergic pathways or neurons or neurotransmitters or receptors can be included in the rotational combination therapy. To prevent adverse side effects associated with administration of multiple therapeutics at once or to target alternative molecular pathways, the protocols can be adjusted to administer fewer medications in combination, and rotate the combinations of therapeutics. h. Combination Therapies to Treat Chronic Obstructive Pulmonary Disease (COPD)
Chronic obstructive pulmonary disease (COPD) is a type of progressive lung disease characterized by shortness of breath and cough. The two classic COPD phenotypes are emphysema and chronic bronchitis. COPD is not curable; a number of medical treatments are used in the management of stable COPD and exacerbations, including bronchodilators, corticosteroids, inhibitors of the enzyme phosphodiesterase-4, and antibiotics. Bronchodilators for treatment of COPD include short-acting beta? agonists [SABAs], long-acting beta? agonists [LABAs], shortacting muscarinic antagonists [SAMAs], and long-acting muscarinic antagonists [LAMAs],
Previous studies have shown that long-term treatment with a combination inhaler containing inhaled corticosteroids and long-acting beta? antagonist is more effective than either agent alone in improving COPD symptoms and in reducing exacerbation frequency. If a dual therapy is not effective, triple therapy with a combination of ICS, LABA and LAMA is indicated. Different, complementary pharmacological mechanisms of action of the three medications improves clinical benefits. For example, a triple fixed-dose combination of extra fine beclomethasone dipropionate (100 pg/puff), formoterol fumarate (6 pg/puff), and glycopyrronium bromide (12.5 pg/puff) administered via a hydrofluoroalkane pressurized metered dose inhaler is superior to fixed ICS/LABA combined therapy and also superior to the LAMA tiotropium for improving lung function and exacerbation prevention in COPD patients at risk of exacerbation.
Thus, there previously are combination therapies used for treatment of diseases, disorders, and conditions, including those described above. Ultimately, this combination will not avoid the problems associated with desensitization to long-term treatment. These combination therapies can be improved by rotating combinations of the drugs. Any of the previously described combination therapies, where there are different targets for therapeutic intervention or can be modified by rotating combinations of therapeutics or treatments to produce a rotational combinatorial therapy regimen described herein. For example, to prevent receptor downregulation upon continuous administration or adverse side effects associated with administration of multiple medications on one day, the protocols can be modified to administer fewer medications in combination, and rotate the combinations of medications. i. Combination Therapies to Treat Obesity-Associated Diseases and Conditions
Rotational combinatorial therapy, such as rotational combinatorial pharmacology provided herein can be used to treat diseases, disorders and conditions that are comorbid with overweight and obesity, including, but not limited to, metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
Combinatorial therapy and rotational combinatorial therapy, such as rotational combinatorial pharmacology provided herein can be used to treat cardiovascular disease. Rotational combinatorial therapy can be used in the treatment of cardiovascular diseases including ischemia reperfusion injury resulting from stroke, myocardial infarction, cardiopulmonary bypass, coronary artery bypass graft, angioplasty, or hemodialysis. Rotational combinatorial therapy also can be used in the treatment of the inflammatory response associated with cardiopulmonary bypass that can contribute to tissue injury. For example, a combination of therapeutics can be administered in accord with a regimen described herein prior to, or in order to prevent cardiovascular disease or an adverse cardiac event. In other examples, a combination(s) of therapeutics can be administered in accord with a regimen described herein subsequent to a cardiac event to prevent further injury from the cardiac event or to aid in recovery from the adverse cardiac event (e.g., heart attack or stroke). In one example, a combination(s) of therapeutics can be administered to a subject in accord with a regimen described herein in order to ameliorate the symptoms of cardiovascular disease or the secondary damage or effects resulting from cardiovascular disease. Amelioration of symptoms can be assessed by methods described herein or those known to the skilled physician. j. Combination Therapies to Treat Overweight and Obesity
There is a lack of therapeutics for obese patients that also focus on polypharmacy for diabetes, metabolic syndrome, and other associated diseases, disorders, and conditions. The approach to weight loss and muscle development described herein is shown to produce results using pharmacological approaches that are at least as effective as bariatric surgery, but without the risks of surgery. This is described in the following sections and detailed in the working examples.
G. COMBINATORIAL AND ROTATIONAL COMBINATORIAL THERAPY FOR WEIGHT LOSS
As described in the next sections, obesity is an exemplary condition for which combinatorial therapies, and rotational combinatorial therapies can be designed. Fat can be stored subcutaneously or as viscerally subcutaneous and visceral adipose tissue. Subcutaneous fat lies underneath the skin and is visible, and is the fat that changes after body composition improves (i.e., fat decrease) with cardiovascular resistance training. Visceral adipose tissue cannot always be seen directly and distributes around the organs, making it more dangerous to health and is more strongly associated with metabolic syndrome and diabetes compared to subcutaneous fat.
Visceral fat actively contributes to health because it produces cytokines and immunoregulatory hormones. Excess of cytokines and immunoregulatory hormones are seen with obesity, causing inflammation and increased risk of cardiovascular disease, immune-dysregulation and a negative effect on cells sensitivity to insulin further contributing to diabetes.
Obesity can induce a series of chronic metabolic diseases, such as diabetes, dyslipidemia, hypertension and nonalcoholic fatty liver disease.
1. Limitations of Existing Treatments for Weight Loss
Given the limitations of lifestyle interventions and bariatric surgery, pharmacotherapeutic approaches for the treatment of obesity are important options. Development of anti-obesity medications has been slow and ineffective. During the past 20 years, several anti-obesity drugs have been discovered, marketed, and subsequently withdrawn from the market; despite showing efficacy during initial stages of treatment, therapeutics for obesity have been accompanied by adverse sideeffects following long-term use.
With the exception of the GLP1R agonist, semaglutide, and the combination medication tirzepatide, which activates the GLP-1 and GIP receptors, the average percent body weight reduction for registered drug treatments varies, but they are in the single-digit range (Bray et al.. Lancet 387: 1947-1956 (2016)). An improved antiobesity treatment(s) is needed to correct excess weight while reducing risk for cardiovascular-associated adverse effects and psychological adverse effects. Likewise, an improved treatment for overweight or obesity can decrease risk for obesity-related comorbidities including metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), cancer, elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others.
Significant and harmful side effects are associated with weight loss therapies. Several limitations on surgery exist, including high cost, and the potential for intraabdominal abscess formation, thrombosis, dehydration, and type 1 diabetic ketoacidosis. As noted above, pharmaceutical weight loss treatments are not as effective as surgical interventions (ie., generally do not show high percent weight loss) and increase the risk of adverse side effects, including nausea, vomiting, headache, constipation, confusion and dizziness, impaired alertness and motor coordination, worsening of depression or suicidal thinking, complex sleep behaviors, sleep paralysis, and compromised respiratory function.
The robust escalations in obesity and associated health complications constitute major driving forces for the discovery of targets and for the development of safe and effective weight loss therapeutics. The combinatorial and rotational therapy provided herein is different from previous approaches for obesity and other disease processes because they are designed with combinatorial and rotational pharmacology (CRP) in mind.
2. Obesity and the Challenges of Treatment
Treatment for obesity can be challenging because of the multitude of causes. A primary treatment for obesity includes dieting and physical exercise. The combination of dieting and exercising, however, rarely produces sustained weight loss, generally resulting from slow weight regain over time.
Altered eating (e.g., dieting) has been recommended as a behavioral change to increase weight loss. CDC recommends a variety of lifestyle interventions for weight loss, including: calorie restriction; time restricted feeding, where meals are consumed within a limited time window (ie., 6-8 hours) during the day; altemate-day fasting, where food consumption is unrestricted every other day and minimal or no calories are consumed on the other days; “5:2” eating patterns, where meals are unrestricted for 5 days each week, followed by 2 days of restricted calorie intake; and periodic fasting, where calorie intake is restricted for several days in a row (ie., 5 days) once per month, and food consumption is unrestricted on the other days.
Increased exercise is recommended as a behavioral change to increase and sustain weight loss. The American College of Sports Medicine (ACSM) and CDC Guidelines propose increasing exercise to augment weight loss. For example, the Guidelines indicate that healthy adults aged 18-65 years should participate in moderate intensity aerobic physical activity a minimum of 30 min on five days per week, or vigorous intensity aerobic activity for a minimum of 20 minutes on three days per week.
Behavioral changes targeting lifestyle changes has been studied extensively, with weight loss typically ranging from 3-10% of body weight with 12 months of intensive treatment in research settings. Nonetheless, most of that weight lost is regained within 12 months of initiating a program. Lifestyle modifications can be used alone, or as an adjunct to medical or surgical treatments for overweight and obesity, and for long-term treatment and management. (Jin (2018) JAMA 320(17): 1210). There are some metabolic and hormonal counterforces that reduce or reverse weight loss; these include an increase in appetite, reduction in energy expenditure, reduced insulin sensitivity that favors growth of adipocyte size and numbers. For example, weight loss is accompanied by persistent endocrine adaptations that cause an increase in appetite and decrease satiety, and there can be a physiological downregulation of pathways associated with weight loss and metabolism, thereby resisting continued weight loss and conspiring against long-term weight maintenance. This can lead to more robust weight regains as the weight lost progresses further from the “weight thermostat” set by years of overeating and all the offsetting mechanisms being activated.
Lifestyle and behavioral modifications are inadequate to provide long lasting weight loss and significant metabolic changes. Pharmacotherapy is necessary in many cases to assist with the metabolic derangements that occur in obese patients but results, as noted below, are very limited. A variety of weight loss treatments have been developed; exemplary therapeutics and treatments are summarized in the following sections.
3. Pharmacological Treatments
Obesity is a chronic degenerative disease that can stem from the rewiring of biological mechanisms that hinder weight loss and promote weight gain. Although lifestyle and behavioral interventions provide moderate efficacy, these strategies are limited by complications in adherence. They can be augmented by surgical intervention and/or pharmacological approaches (Muller TD. et al. Nat Rev Drug Discov 21(3):201-223 (2021)). Bariatric surgery generally is considered the most effective approach to weight loss, but it is expensive and limited to single patients, lacking the reach necessary to treat obesity at a global scale. Pharmacological agents can offer such treatment.
The historical development of anti-obesity medications is challenging because of the limited understanding of the molecular mechanisms that control appetite and adverse effects. Historical anti-obesity drug classes include mitochondrial uncouplers, amphetamines, thyroid hormones, drug cocktails, neuromodulators, lipase inhibitors, cannabinoid receptor antagonists, and gastrointestinal-derived peptides chemically optimized for pharmaceutical use (Muller TD. et al. Nat Rev Drug Discov 21(3):201- 223 (2021)). Therapeutic approaches regulate the function of pathways related to energy balance and systemic energy sensing. Mechanisms of action can involve limiting fat absorption or food intake, increase satiety, or facilitate energy expenditure.
Medical standard of care for weight loss focuses on pharmacological monotherapies and occasionally dual therapies. FDA-approved monotherapy options include phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phentermine-topiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave®) and semaglutide (sold under the trademark Wegovy®) medications. These medications, and others, can target components of the central nervous system or peripherally, such as in the gastrointestinal system, including, but not limited to the stomach, small intestine and colon, and pancreas (see e.g., FIG. 2).
As described and exemplified herein, monotherapies have limited efficacy, in part due to the metabolic redundancies and recruitment of alternate and counter- regulatory pathways, and desensitization. Obesity is a disease with multiple etiologies; as described herein can be treated with a multi -targeted approach. As exemplified and described herein, a multi-target approach provides greater benefit than any single medication alone. A multi-targeted approach includes a combination therapy or a rotational combinatorial therapy.
A number of medications have been administered for long-term use for weight loss; these medications, include, but are not limited to, phentermine-topiramate, orlistat, lorcaserin, naltrexone-bupropion, and liraglutide. Treatment with these medications as monotherapies results in weight loss after one year that ranges from 3.0 to 6.7 kg (6.6-14.8 lbs) compared to placebo (Heymsfield et al., (2017) The New England Journal of Medicine 376 (3):254- 266). Information on how these drugs affect longer-term complications of obesity, such as cardiovascular disease or death is sparse. Obesity drugs do not target weight loss per se, but on appetite suppression and/or are label expansions of other marketed drugs. As a result, treated subjects have significant adverse side effects, and the medications have relatively poor efficacy. For example, semaglutide (sold under the trademark Wegovy®) medication is a therapy designed for treatment of type-2 diabetes. An effect of the drug was weight loss and it has been rebranded for weight loss treatment.
The following are exemplary weight loss drugs that can be combined as described herein for combination therapy and also in a rotational combinatorial therapy regimen for treating overweight, obesity, for effecting weight loss and for treating secondary complications of each of the preceding conditions. The combinatorial therapy can include a combination of therapeutics known to effect weight loss and/or pharmaceuticals previously used as weight loss monotherapies and dual therapies. Medications that previously were used for non-weight loss indications that exhibit weight loss as a secondary effect also can be included in the combination therapies herein. The medications listed herein, such as, for example, below can be included in the combinations herein for treating overweight, obesity and/or for weight loss. Pathways associated with satiation, metabolism, hunger and weight gain or loss can be identified and used to formulate targets and/or therapeutics for the combination therapy, such as therapeutics listed below. Combinatorial regimens employing combinations, such as three or more of the medications below can be included in the combinations for weight loss, and/or treatment of overweight or obesity. These drugs can be part of a rotational combinatorial regimen as well, in which two or more of the drugs are combined and administered for a predetermined time, and then are rotated with another drug or combination of drug for a predetermined time. a. Amphetamines (e.g., phentermine-topiramate)
Amphetamines stimulate norepinephrine release that can result in increased blood pressure, heart rate, and cardiac excitability via binding to vasculature and heart adrenergic receptors. These drugs belong to the class of drugs known as sympathomimetic amines. Amphetamines used as anti-obesity medication include, but are not limited to Methamphetamine (Desoxy ephedrine), Phenmetrazine (sold under the trademark Preludin®), Phendimetrazine, Phentermine, benzphetamine and Diethylpropion. Phentermine (sold under the trademark Adipex-P®) and Diethylpropion (sold under the trademark Tenuate®) were generally designed to retain anorectic activity, but with reduced effects on the cardiovascular and brain reward system (Colman, Ann., Intern. Med. 143, 380-385 (2005)). Several clinical studies report the absence of major adverse effects of phentermine or diethylpropion on blood pressure and heart rate; nonetheless, their use is contraindicated in patients with hypertension or elevated risk for cardiovascular disease (Muller etal., Nat Rev DrugDiscov 21 : 201-223 (2022)). Patients also quickly develop a tolerance to these medications.
Phentermine is a weight loss medication approved for short term use, such as less than a month (Hendricks et al., Obesity 17: 1730-35 (2009)). Patients can rapidly develop a tolerance to the medication. Amphetamines can be used in combination with other medications for treatment of overweight and obesity. For example, phentermine is used in combination with topiramate (combination sold under the brand name Qsymia®), which can be used for a longer period of time than phentermine alone. Previous studies show that the combination therapy results in an average of 5-10% weight loss, where weight loss corresponds to dosage. These can be administered in combination with liposomes provided herein, or incorporated into the liposomes. b. Lipase inhibitors (e.g., Orlistat)
Lipases are a class of digestive enzymes produced in the pancreas, mouth, and stomach to metabolize fat and to facilitate nutrient uptake. Orlistat (sold under the trade name Xenical®) is a lipase inhibitor that reduces the uptake of dietary fat in the gastrointestinal tract. The resulting fat malabsorption after treatment with lipases facilitates a negative energy state leading to a placebo-normalized weight loss in the range of 2.5%. Orlistat has shown beneficial effects on blood glucose, non-alcoholic fatty liver disease, and blood pressure (Muller et al. (2018) Pharmacol. Rev. 70:712- 746; Khera et al. (2018) Gastroenterology 154'.1309-1319. e7). The most common adverse events after orlistat treatment are of a gastrointestinal or digestive nature. c. Serotonergic agonists - Neuromodulators (e.g., lorcaserin)
Serotonergic agonists act as anti-obesity medication by suppressing appetite through the activation of serotonin receptors. Clinical serotonergic agonists include Fenfluramine, Dexfenfluramine, Sibutramine, Lorcaserin (sold under the trademark Belviq®), and Tesofensine. Cardiovascular safety concerns accompany Fenfluramine, Dexfenfluramine, and Sibutramine (Muller et al. (2018) Pharmacol. Rev.70 712- 746). The FDA requested withdrawal of Lorcaserin due to clinical trials showing an increased occurrence of cancer (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). Tesofensine, an inhibitor of norepinephrine, serotonin, and dopamine reuptake that was initially advanced for treatment of Alzheimer disease. A phase II study, it was reported to dose-dependently decrease body weight by 4.4-10.4% (Wharton et al. (2015) J. Curr. Cardiol. Rep. 17:35,' Astrup A. et al. (2008) Lancet 372:1906-1913). d. Bupropion/Naltrexone (such as the product sold under the trademark Contrave®) Naltrexone, an opioid antagonist, blocks the inhibitory effects of opioid receptors activated by P-endorphin released in the hypothalamus, which stimulates feeding. Naltrexone reduces food intake in combination with bupropion, reuptake inhibitor of dopamine and norepinephrine. Naltrexone does not cause weight loss in monotherapy; patients treated with Naltrexone 32 mg plus Bupropion showed a body weight reduction of 6.1% after 56 weeks of treatment (Greenway et al. Lancet 376, 595-605 (2010)). No increased adverse cardiovascular events were noted in an analysis of a cardiovascular outcome trial after bupropion/naltrexone treatment (Nissen et al., JAMA 315:990-1004 (2016)). Another study showed that addition of a modified diet and increased exercise to the bupropion/naltrexone combination lost an average of 9.3% of their baseline body weight. e. Glucagon-like peptide-1 receptor (GLP1R) agonists
Glucagon-like peptide-1 (GLP1; SEQ ID NO: 1) acts at the pancreas to enhance the expression and secretion of insulin and to inhibit the release or glucagon. GLP1 and glucose-dependent insulinotropic polypeptide (GIP; SEQ ID NO: 10) are primary incretin hormones secreted from the intestine on ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic P cells. Although the specific mechanism of action is multifactorial, with gut, brain, and systemic improvements in insulin sensitivity, GLPR1 antagonism can lead to decreased body weight via the inhibition of food intake (Muller et al. (2018) Pharmacol. Rev. 70:712-746).
Chronic treatment with GLP-1 receptor agonists causes weight loss in diabetic humans. Liraglutide (sold under the trademark Saxenda®), is a once-daily injectable GLPR1 agonist, and was the first GLP1R agonist approved for treatment of obesity. Liraglutide is short-acting, so it is administered daily. After one year of treatment, there was a reported mean decrease of 8% body weight in subjects treated with liraglutide compared to 2.6% in subjects treated with vehicle controls (Pi-Sunyer et al. (2015) N. Engl. J. Med. 373: 11-22). In 2020, treatment with 3 mg liraglutide was approved for weight management in adolescents with obesity.
GLP-1 increases insulin metabolism and plays a role in appetite and digestion. It is among the incretins, which are hormones released by the small intestine into the bloodstream following a meal to help lower blood sugar by triggering insulin and blocking other sugar sources. Medications that are GLP-1 agonists are referred to as incretin mimetics since they “mimic” the incretin hormone effects.
Semaglutide, which is similar to and acts like a GLP-1 hormone, is used as an anti-diabetic medication. Semaglutide also slows down the rate at which food leaves the stomach (called gastric emptying). These actions cause a feeling of fullness, lowering appetite and resulting in weight loss. Semaglutide (sold under the brand name Wegovy®) is an injectable GLPR1 agonist FDA-approved in 2021 for chronic weight management in adults with obesity (URL: fda.gov/news-events/press- announcements/fda-approves-new-drug-treatment-chronic-weight-management-first- 2014). Wegovy® weight loss medication is approved for treatment of adults with a body mass index (BMI) greater than or equal to 30mg/kg2 alone or 27 mg/kg2 with at least one weight-related comorbidity (e.g., high blood pressure, high cholesterol). Semaglutide administered at a dose of 2.4 mg lowers mean body weight to -12-15% after 68 weeks of treatment (relative to -2.4% in placebo controls) with once-weekly treatment (Wilding et al., N. Engl. J. Med. 384(11): 989-1002 (2021)). A longer mechanism of action compared to other weight loss medications allows semaglutide to be administered weekly; semaglutide is the first once-weekly GLP-1 agonist approved for weight loss. Daily dosing of 2.4 mg achieved >15% weight loss in half of the study participants, whereas one third experienced more than a 20% reduction (O’Neil et al., Lancet 392:637-649 (2018)). Semaglutide is well-tolerated although the adverse effects (nausea, diarrhea, vomiting and constipation) typified by GLP1- related remain. The high level of side effects can limit a patient’s ability to maintain or reach a target level of the drug. In such cases, a provider can extend the period to reach the target dose, or may have to discontinue treatment if the patient is unable to tolerate the target dose, due to the adverse side effects.
Several peptide and small-molecule GLP1R agonists are in clinical development, including formulations designed for oral administration (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). GLP1R agonists in clinical development include Efpeglenatide (s a long-acting GLP-1RA that is a single amino acid-modified exendin conjugated to a fragment crystallizable region of human immunoglobulin 4 via a 3.4-kDa mini-poly ethylene glycol linker), semaglutide sold under the trademark Rybelsus®, Danuglipron (formerly PF-06882961), GLPR-NPA, and lotiglipron (formerly PF-07081532). Several GLP-1 receptor agonists approved to improve glycemic control and lower blood sugar in adults with type 2 diabetes exhibit a secondary effect of increased weight loss. For example, type 2 diabetes patients taking >1500 mg/day metformin in combination with the GLP-1 receptor agonist sold under the tradename dulaglutide, sold under the tradename Trulicity®, at 1.5 mg, 3.0 mg, or 4.5 mg once weekly lost an average of 6.6, 8.4 and 1.1 pounds, respectively, after 36 weeks on the medication. f. Mitochondrial uncouplers
An alternative approach to decreasing food intake and/or absorption is to increase the metabolic efficiency by which food is converted into useful energy. Highly thermogenic tissues such as brown adipose tissue show high expression of Uncoupling Protein 1 (UCP1, SEQ ID NO:41) that uncouples oxygen consumption from ATP synthesis. Pharmacological upregulation of UCP1 activity can be induced by catecholamines or small molecule uncouplers. Small molecule uncouplers for antiobesity application include 2,4-dinitrophenol (DNP) and BAM15 ((2- fhiorophenyl){ 6-[(2-fluorophenyl)amino]( 1 ,2, 5 -oxadiazolo [3 ,4e] pyrazin-5-yl) } amine). DNP is the first small molecule uncoupler tested clinically, but has limited utility owing to toxicity. Controlled-release oral formulation of DNP are under investigation to achieve an enhanced therapeutic index (Muller el al.. Nat Rev Drug Discov 21 : 201-223 (2022)). BAM15 is an orally administered therapeutic that reverses diet-induced obesity and insulin resistance in mice (Alexopoulos et al., Nat. Commun. 11(1): 2397 (2020)). g. Thyroid hormones
Thyroid hormone can decrease body weight and body fat by stimulating energy expenditure (Muller et al., Pharmacol. Rev. 70:712-746 (2018)). Thyroid hormone also can improve hepatic lipid metabolism and decrease low-density lipoprotein (LDL) cholesterol via enhanced reverse cholesterol transport and clearance of LDL via the liver (Baxter et al., Nat Rev Drug Discov 8:308-320 (2009)). The biologically active form of thyroid hormone is tri-iodothyronine (T3), which promotes its pharmacology through two specific nuclear thyroid receptor (TR) isoforms, TRa and TRp. Administration of T3 increases metabolic rate in a variety of species, including mice, rats, and humans (Muller et al., Pharmacol. Rev. 70, 712-746 (2018)). The molecular mechanism underlying T3 modulation of metabolic rate includes uncoupling of oxidative phosphorylation from mitochondrial ATP synthesis in skeletal muscle and other peripheral tissues, regulation of lipogenesis, activation of Na+/K+ ATPase, enhanced mitochondrial biogenesis, and stimulation of futile cycling (Muller el al.. Pharmacol. Rev. 70:712-746 (2018)). Excess thyroid hormone also leads to muscle and bone catabolism, as well as several cardiovascular adverse effects including cardiac arrhythmia, tachycardia, and heart failure, severely limiting its use as a weight loss therapeutic. h. Drug cocktails
Metabolic redundancies and recruitment of alternate and counter-regulatory pathways can limit the efficacy of monotherapies. Clarkotabs, developed in 1941, were among the first commercially distributed combination diet pills; the combination sought to harness the anorectic effect of amphetamines with the thermogenic effect of thyroid hormone, with Aloin and Atropine sulfate to counteract potential adverse cardiovascular effects (Muller et aL, Pharmacol. Rev. 70:712-746 (2018)). Later combinations included cocktails of weight-reducing substances, including d- amphetamine or related analogs (e.g., Diethylpropion, Fenfluramine, Sibutramin, or Fenproporex), thyroid hormones, diuretics, laxatives, Chlorthalidon, Ephedrine, and/or Phenolphthalein. Substances such as Digitalis, Belladonna, benzodiazepines, barbiturates, corticosteroids, cardiac glycosides, beta-blocker, and potassium were common additives used to counteract or mask adverse cardiovascular effects of the drug cocktail (Cohen PA. et al. , Am J Public Health 102: 1676-1686 (2012)).
The combination of the amphetamine phentermine and topiramate was approved by the FDA in 2012. Topiramate is a sulfamate-substituted monosaccharide derived from D-fructose, commonly used to treat epilepsy and migraine. Although the mechanism of how the combination improves systemic metabolism is unknown, placebo-normalized weight loss ranges from 5.9%-9.6% (Muller et al., Pharmacol. Rev. 70:712-746 (2018)).
The weight loss combination of the medications naltrexone and bupropion combines two medications approved for indications other than weight loss. Bupropion, a dopamine-norepinephrine reuptake inhibitor is approved to treat depression and aid in smoking cessation, and naltrexone, an opioid receptor antagonist is approved to treat alcohol and opioid dependence. The naltrexonebupropion combination is marketed under the trade name Contrave® and is FDA approved for chronic weight management. i. Cannabinoid receptor antagonists
Rimonabant, an endocannabinoid 1 receptor (CB1) antagonist, acts by modulating neurons in both homeostatic and hedonic feeding circuits, leading to placebo- subtracted weight loss of ~2.6-6.3 kg (Muller etal., Pharmacol. Rev. 70: 712-746 (2018); Pi-Sunyer et al. JAMA 295:761-775 (2006)). Rimonabant was discontinued in 2009 due to serious adverse psychiatric effects (Sam et al., J Obes 2011 :432607 (2011)). j. GIPR agonists and GIPR/GLP1R combination agonists
Glucose-dependent insulinotropic polypeptide (GIP; SEQ ID NO: 10) is a hormone involved in blood sugar control. GIP receptor (GIPR) agonists have been shown to decrease food intake, increase energy expenditure, decrease body weight and to improve glucose handling in preclinical studies (Mroz et al., Mol. Metab. 20:51-62 (2019); Zhang et al., Cell Metab. 33:833-844.e5 (2021)). GIP is known to be upregulated following Rou-en-Y gastric bypass.
Body weight loss associated with GLP-1 agonist treatment is enhanced when GLP-1 and GIP are co-administered (Matthias Tschbp oral presentation at ADA (American Diabetes Association), 2011; Tschbp et al., Diabetes 66: 1766-1769 (2017)). GIPR/GLP1R dual agonists and co-admini strati on of GLP-1R agonists and GIPR agonists have demonstrated metabolic benefits and reduced body weight in mice when compared to GLP1R agonists (Coskun et al., Mol. Metab. 18: 3-14 (2018); Finan et al., Sci. TranslMed. 5, 209ral51 (2013)). Thus, in addition to improving blood glucose control, GIP can also enhance GLP-1 -mediated body weight loss. GIP/GLP1 dual agonists include Tirzepatide, GIP/GLP peptide I, GIP/GLP peptide II, and NN9709.
Tirzepatide is a combination medication that activates the GLP-1 and GIP receptors; tirzepatide contains GLP-R and GIPR agonists). Tirzepatide (available under the trademark Mounjaro®) is a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist. The drug is manufactured by Eli Lilly & Co. and was approved in May 2022. Tirzepatide works similarly to the other drugs in the GLP-1 receptor agonist family, but it has additional effects that appears to give it a slight edge. It has a dual-action design, mimicking the action of two incretin hormones involved in blood glucose control: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This is the first-in-class medicine to act on both of these receptors.
Tirzepatide is a 39 amino acid peptide GLP-1 receptor agonist based on the GIP sequence. It contains 2 noncoded amino acids in positions 2 and 13, a C-terminal amide, and a lysine residue at position 20 that is attached to a 1, 20 eicosanoic acid via a linker. Studies of tirzepatide were conducted through two placebo-controlled trials (SURP ASS-1 and -5), three trials in combination with metformin, sulfonylureas and/or SGLT2 inhibitors (SURPASS-2, -3, -4), and two additional trials conducted in Japan. A phase III trial of tirzepatide in patients with type 2 diabetes of excess weight revealed a body weight reduction of >15% relative to 9% in patients treated with Img semaglutide (Frias etal., N. Engl. J. Med. 385, 503-515 (2021); URL: fda.gov/news- events/press-announcements/fda-approves-novel-dual-targeted-treatment-type-2- diabetes; published May 13, 2022).
A separate study looking at efficacy of tirzepatide (5 mg, 10 mg, 15mg) was conducted via the SURMONT-1 clinical trial, where participants received once a week GLP-1 receptor agonist. Patients receiving the GLP-1 receptor agonist lost up to 22.5% (52 pounds or 24 kg) of their body weight. The study enrolled 2,539 participants across the US, Argentina, Brazil, China, India, Japan, Mexico, Russia, and Taiwan and was the first phase 3 global registration trial evaluating the efficacy and safety of tirzepatide in adults with obesity, or overweight with at least one comorbidity, who do not have diabetes. Study participants were assessed for mean percent change in body weight at week 72 and the percentage of patients achieving >5% weight loss from baseline to week 72 as primary end points. Secondary end points included the percentage of patients achieving greater than or at 10 and 20% weight loss from baseline to week 72. The study results are set forth in the table below:
The study results show that participants taking tirzepatide achieved average weight reductions of 16.0% (35 lb. or 16 kg on 5 mg), 21.4% (49 lb. or 22 kg on 10 mg) and 22.5% (52 lb. or 24 kg on 15 mg), compared to placebo (2.4%, 5 lb. or 2 kg). Additionally, 89% (5 mg) and 96% (10 mg and 15 mg) of people taking tirzepatide achieved at least 5% body weight reductions compared to 28% of those taking placebo. Tirzepatide met both co-primary endpoints of superior mean percent change in body weight from baseline and greater percentage of participants achieving body weight reductions of at least 5% compared to placebo. The study also achieved all key secondary endpoints at 72 weeks. There was no data on actual fat loss or muscle mass loss or retention. Safety and adverse reactions also were assessed; the investigators found that 18%, 17%, 9%, 7% and 5% of trial participants experienced nausea, diarrhea, vomiting, constipation, and abdominal pain, respectively.
Tirzepatide (sold under the trademark Mounjaro®) was approved by the US Food and Drug Administration as a once-weekly subcutaneous injection to treat adults with type 2 diabetes, to improve blood sugar levels. As detailed above, and herein, Tirzepatide showed significantly better long-term blood sugar control (i.e., A1C) and weight loss compared to semaglutide.
Glucagon and glucagon agonists have been shown to promote satiety and to increase energy expenditure when used in combination in both rodents and human (Kleinert et al., Int J Mol Set. 20(21): 5407 (2019)). A triagonist peptide that act as an agonist of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors was previously described to effect weight loss in animal models (Finan et al., Nat Med 21(l):27-36 (2015)). k. GLPIR/glucagon dual agonists
GLP-1 receptor (GLP1R) agonists in combination with glucagon (GcgR) agonists participate in several mechanisms of body weight reduction, including appetite suppression, thermogenesis and lipolysis, while minimizing the risk of hyperglycemia (Muller et al., Nat Rev Drug Discov 21 (3):201 -223 (2021)). Combination strategies of these agonists have included Cotadutide (MEDI0382), BI 456906, Efinopegdutide (LAPSGLP/GCG), and oxyntomodulin (OXM; SEQ ID NO:4). Several GIP/GLP1 /glucagon tri-agonists also have been developed for weight loss, including HM15211 (LAPSTriple Agonist), GGG tri-agonist, LY3298176 and NN9423.
1. Summary
With few exceptions, the field of pharmaceutically assisted weight loss has focused on monotherapy and the most effective pharmaceuticals include versions of a GLP-1 agonist. Although approved for use in human, several therapeutics that show efficacy are not widely used because of adverse side effects or toxicity associated with administration of the medications at the therapeutic or FDA approved dosages. For example, patients taking Rimonabant experience adverse psychiatric effects, patients taking thyroid hormones and amphetamines experience adverse cardiac effects, patients taking lorcaserin show increased occurrence of cancer, and patients taking orlistat experience gastrointestinal issues.
A combination of therapeutics administered in accord with the rotational protocol/regimen described herein incorporates pharmacological principles known to occur after a gastric bypass, as well as overcoming tolerance and/or desensitization to medications due to receptor downregulation/desensitization to medications. The combination therapy can be administered at a lower dosage than the monotherapeutic dosage to decrease side effects associated with administration of the monotherapy. The combination therapy is rotated at intervals (ie., monthly, bi-monthly, or trimonthly) wherein the patient is not administered an individual therapeutic for an extended period; the finite time period for administration of the medication decreases the time for developing adverse side effects (z.e., severe adverse effects) or downregulation of the pathways activated by the medications.
The rotational combinatorial therapy described herein can include a combination of a plurality of different known therapeutics and/or treatments. For example, the combination for treatment of obesity or for weight loss can include more than one previously characterized weight loss treatment. In some examples, the rotational combinatorial therapy can include one or more FDA-approved monotherapy options, such as, for example, phentermine (sold under the trademark Adipex-P®), orlistat (sold under the trademark Xenical®), lorcaserin (sold under the trademark Belviq®), liraglutide (sold under the trademark Saxenda®), phenterminetopiramate (sold under the trademark Qysmia®), naltrexone-bupropion (sold under the trademark Contrave ®), tirzepatide (sold under the trademark Mounjaro®), and semaglutide (sold under the trademark Wegovy®) medications. The rotational combinatorial therapy provided herein also can be used for treatment of any condition with a previously characterized mechanism of action or pathway, where a combination of therapeutics can be developed for treating the disease or disorder or condition. For example, a treatment of obesity or overweight, for which a plurality of different known molecular targets for treatment or multiple known molecular or cellular pathways are known to be involved in disease progression; a combinatorial therapy including one or more of amphetamines, lipase inhibitors, serotonergic agonists, opioid agonists, Glucagon-like peptide-1 receptor (GLP1R) agonists, mitochondrial uncouplers, thyroid hormones, diuretics, dopamine-norepinephrine reuptake inhibitors, cannabinoid receptor antagonists, GIP agonists, GIPR agonists, and GLPIR/glucagon dual agonists can be administered in a rotational combinatorial therapy to target multiple pathways that are associated with a disease or disorder, but where a pharmacotherapy has not yet been developed.
4. Surgical Treatments
The most effective treatment for obesity has been bariatric surgery (Kissler et al., (2013) Semin Nephrol. 33(l):75-89). Bariatric procedures include, for example, laparoscopic adjustable gastric banding, Roux-en-Y gastric bypass, vertical-sleeve gastrectomy, and biliopancreatic diversion. Surgery for severe obesity is associated with long-term weight loss, improvement in obesity -related conditions, and decreased overall mortality. A resolution of co-morbidities, such as diabetes, hypertension, fatty liver, urinary distress, and hyperthyroidism, also have been shown to occur following bariatric surgery (Susmallian et al., Medicine 98(3): el3824 (2019)).
Weight loss of between 12% and 35% (depending on the type of procedure performed) is expected at 1-2 years after surgery. The average weight loss in clinical practice after 2 years for adjustable gastric banding is about 20%, weight loss after Roux-en-Y gastric bypass is about 30%, and weight loss after biliopancreatic diversion or duodenal switch is about 35% (Cummings et al., (2004) J Clin Endocrinol Metab 59:2608-2615). Complications occur in about 17% of cases and reoperation is needed in 7% of cases (Chang etal., JAMA Surgery (Meta-analysis, Review) 149 (3): 275- 87 (2014); doi: 10.1001/jamasurg.2013.3654). Weight loss after bariatric surgery results, not only from the bypass and other physical changes, but also from factors other than the physical changes to the gastrointestinal tract and calorie reduction following surgery (Cummings et al. , J Clin Endocrinol Metab 89:2608-2615 (2004)). Post-surgical hormonal alterations occur that can change activation of brain pathways that regulate appetite cues (Batterham et al., Nature 450: 106-109 (2007)).
Vagal nerve stimulation (VNS) delivers short bursts of energy into the vagus nerve in the brain, is used for treatment of drug-resistant epilepsy, and has been associated with weight loss. VNS has been associated with weight loss (> 5% of body weight) within 6-12 months of stimulation. The weight loss is due to the action of afferent fibers of the nerve which participate in the brain-gut axis, which take part in a feedback loop induced by the presence of food in the gastrointestinal tract inducing hypometabolism of the hypothalamus and subsequent involvement of the satiety centers, thereby controlling food intake.
Because of the high monetary costs of surgery, the extensive and taxing lifestyle modifications, and substantial risks, effective and less invasive treatments are needed. As described herein, the benefits of surgical intervention, can be achieved by applying the combinatorial, and rotational combinatorial methods provided and described herein for weight loss by selecting and administering combinations of drugs/treatments to mimic the hormonal and physiological effects of surgery. The combinations of therapeutics also can be rotated.
Exemplary Small molecules drugs for use for weight loss and other indications are set forth in the following table: These small molecule drugs can be formulated as part of the liposomes (inside and outside) or administered as a separate dosage from the liposomes provided herein, where the liposomes display and/or contain one or more peptide drugs.
H. MODIFICATIONS AND ENHANCEMENTS OF PHARMACOLOGICAL WEIGHT LOSS TREATMENTS TO IMPROVE CLINICAL OUTCOMES
Complex and redundant systems control food intake and energy expenditure (see Lenard et aL, Obesity (Silver Spring) 16 (Suppl 3): SI 1-22 (2008). Pharmacotherapy treatments and candidates under clinical development for treatment of overweight and obesity can be characterized, for example, as follows: 1) medications that act peripherally to impaired dietary fat absorption; 2) medications that act centrally to decrease food intake; and 3) medications to facilitate energy. Medications that increase muscle development also are used. Despite compliance with weight loss guidelines, many drugs launched for the treatment of obesity over the last two decades have been withdrawn due to safety issues associated with increased risk of cardiovascular or psychiatric complications. The failure of these medications can be attributed to the multifactorial pathogenesis and the complex neurohormonal regulation of energy balance. Although monotherapy provides some efficacy for treatment of a disease, disorder, or condition where the target is a single protein in a pathway involved in obesity, physiological counter-regulatory mechanisms involving alternate pathways pose imitations. Consequently, as described herein a disease with multiple targets and/or that involves multiple pathways and/or etiologies requires a multi -targeted approach. Obesity is exemplary of such diseases, disorders, and conditions in which multiple pathways are involved. This approach can be applied to other chronic diseases, disorders, and conditions that involve a plurality of targets/pathways/etiologies. These methods also are for treating diseases, disorders, and conditions for which the subject becomes desensitized to treatment, requiring higher doses and consequent increased risk of adverse effects.
As exemplified by treatment of obesity, the combinatorial therapy and rotational combinatorial therapies provided herein correct excess weight while reducing risk for adverse effects (z.e., cardiovascular and psychological adverse effects) and without increasing muscle loss by taking into account the multifactorial processes associated with metabolism and weight gain. Combinations for use in the combinatorial and rotational combinatorial therapies provided herein include a plurality of medications and/or treatments. The combinations can be rotated to overcome downregulation that occurs after prolonged monotherapy. The combinations target the multifactorial processes involved in weight gain or failure to lose weight. The rotation prevents or mitigates the desensitization/downregulation that occur upon long-term exposure to some of the weight loss therapeutics. The rotational combinatorial therapy herein includes combinations of medications that: act peripherally to impaired dietary fat absorption; act centrally to decrease food intake; facilitate energy expenditure; and increase muscle development.
The combinatorial, and the rotational combinatorial therapies described herein for treating overweight or obesity also can decrease risk for obesity-related comorbidities including metabolic syndrome, obstructive sleep apnea, non-alcoholic fatty liver disease, diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (e.g., heart attack, stroke), cancer, elevated blood pressure, elevated blood cholesterol, and elevated triglyceride levels, and others. As a non-surgical option for treating obesity and overweight, the combination and rotational combinatorial therapy described herein is not associated with the harmful side effects associated with surgery, such as high cost, and the risk of developing intra-abdominal abscess, thrombosis, dehydration, and type 1 diabetic ketoacidosis.
1. Developing Combination and Rotational Combinatorial Therapies for Weight Loss
Developing a combinatorial therapy and rotational combinatorial therapy for weight loss, such as for use for treating overweight or obesity, includes assessing a variety of factors, selecting patients for treatment, selecting molecular and/or cellular pathways for treatment, selecting therapeutics for treatment, and developing the rotational regimen, including timing for administration and timing for rotating the combinations, and dosages. For example, molecular and/or cellular pathways known to contribute to weight loss or appetite regulation or metabolism, or molecular and/or cellular pathways that are associated with weight loss or appetite regulation or metabolism can be targeted for therapy. For example, peptides or proteins that are associated with molecular and/or cellular pathways that contribute to or impact weight loss, appetite regulation, and/or metabolism can be selected for use for treatment of overweight or obesity, or to effect weight loss. The combinations of treatments are identified based on these criteria and those discussed below to combine treatments that, for example, mimic the effects of bariatric surgery. The combinations (or clusters) can be rotated to produce rotational combinatorial protocols to avoid desensitization and/or receptor downregulation.
Several factors can be assessed for developing combinatorial and rotational combinatorial therapies for weight loss. For example, peptides known to contribute to weight loss and/or appetite regulation and/or metabolism can be assessed and included in the combinations. In determining pathways to target for weight loss and peptides to include in the combinations, the following can be assessed: 1) the pharmacodynamic and pharmacokinetic properties of each peptide individually and in combinations with other peptides and/or other therapeutics; 2) stability in serum, to ensure adequate stability for the intended use; 4) solubility of the peptides and/or other therapeutics; the delivery profile to ensure activity, stability, solubility and other characteristics to ensure therapeutic activity and efficacy.
2. Pathways to Target for Weight Loss and Exemplary Polypeptides
Research on new signaling molecules has substantially increased the knowledge of central and peripheral mechanisms underlying homeostatic energy balance. Homeostatic mechanisms involve multiple components including neuronal circuits, some originating in hypothalamus and brain stem, as well as peripherally derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components can result in or contribute to obesity.
Studies of gastric bypass patients show that the surgical procedure, where the stomach is reduced in size and consequent caloric restriction and malabsorption, is not solely responsible for the subsequent weight loss; (see, e.g., Vincent and Roux (2008) Clinical Endocrinology 69(2): 173-179; Komer et al., Obesity 14:1553-1561 (2006)). For example, after a form of gastric bypass surgery, Roux-en-Y gastric bypass (RYGB), 84% of patients who had type-2 diabetes showed complete remission of this disease, and almost all had improved glycemic control. Type 2 diabetes symptoms resolve quickly after surgery and before weight loss, and patients exhibit improved glucose homeostasis after RYGB compared to similar weight loss achieved via other means (z.e., diet and/or exercise). These rapid changes indicate that the resultant weight loss and/or reduced caloric intake, does not explain these weight-independent antidiabetic actions of RYGB.
Post-surgery changes in several of these pathways and others, including gastro-intestinal hormones, including increases in GLP-1, PYY, and oxyntomodulin, decreases in GIP and ghrelin, and a combination of these changes modulate initial and long-term maintenance of weight loss (see e.g., Ochner et al., IntJ Obes 35(2): 153- 166 (2011)). Hormone profiling of subjects after bariatric surgery provides the following insights: 1) weight loss is an integrated hormonal process, 2) multiple peptides are involved (Combination) and 3) peptides have fluctuations and in timing and release (Rotation).
Peptide levels that change after gastric bypass contribute to weight loss following the surgery. In accord with methods herein, peptides and/or other therapeutics, including peptide/receptor agonist or antagonists, depending on the target, in combinations that mimic the fluctuations of peptides whose expression or activity is altered after gastric bypass or other weight loss surgery. The combination therapy herein takes into account the integrated processes that occur during weight loss, and seek to mimic the multiple pathways using combinations of therapeutics administered to mimic the fluctuations in timing and release that occur following gastric bypass. A combinatorial therapeutic regimen is described because multiple pathways are involved and because monotherapies are generally ineffective or less effective than gastric bypass. The combination therapies described herein administered with a rotational approach overcome desensitization to the peptides and/or therapeutics that can occur due to receptor downregulation, which occurs over time after monotherapy or long-term combination therapy.
Weight loss surgery patients also experience an increase in muscle mass following surgery; therapeutic agents that alter pathways that affect (z.e., increase) muscle generation to increase muscle mass during the weight (fat) loss protocol. Weight loss surgery patients also experience a decrease in muscle mass following surgery, and it is provided herein to provide such patients with agents that alter pathways that affect (z.e., increase) muscle generation to increase muscle mass during the weight (fat) loss protocol. Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes. The therapy described herein can include peptides to decrease insulin resistance that can be a comorbidity with overweight or obesity.
For example, in obesity, more than over 20 peptides and hormones are known to orchestrate the cellular milieu. In any of the examples of the combination and rotational combinatorial therapies provided herein the combinations can include peptides that are associated with metabolism, satiety, physiological response to hunger or food ingestion, and/or digestion. These combinations include peptides or therapeutics that target pathways altered following bariatric surgery. The combinations can include therapeutics that impact homeostatic energy balance, including neuronal circuits, some originating in the hypothalamus and brain stem, as well as peripherally derived satiety, hunger and adiposity signals that modulate neural activity and regulate eating behavior. Dysregulation of one or more of these homeostatic components results in obesity. The hormonal signaling network that provides the brain input related to metabolic status and energy stores includes leptin, insulin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), peptide YY3-36 (PYY3-36), and ghrelin. Any of the peptides, proteins, and pathways detailed below can be targeted for the combination and/or combination rotational therapy described herein. The combinatorial therapies and rotational combinatorial therapies provided herein target a plurality thereof. Components of these pathways, including agonists and antagonists as appropriate, or down- or up-regulators of the pathways can be included in the combination and/or rotational combinatorial therapy to effect weight loss, or to treat or ameliorate comorbidities of overweight or obesity.
The following discussion provides and describes exemplary therapeutics that can be incorporated into a combinatorial weight loss protocol. The combinations can be rotated to produce rotational combinatorial protocols.
Glucagon like peptide-1
The sequence of GLP-1 is set forth in SEQ ID NO: 1, and the chemical formula is C186H275N51O59. The glucagon-like peptide-1 (GLP-1) is released by L- cells in response to food ingestion. Simulation of L-cells increases both GLP-1 and GLP-1 related peptides, such as glicentin, oxyntomodulin intervening peptide-2 and GLP-2. Agents described for use in the rotational combinatorial treatment herein can work by activating the Glucagon-like peptide- 1 receptor (GLP-1R) or by inhibiting the breakdown of GLP-1 like dipeptidyl peptidase- 4 (DPP-4) inhibitors (see Bruton, ., Int. J of Clin Pract. 68(5):557-567 (2014)). DPP-1 inhibitors and their uses are previously described (see e.g., WO 2002/068420, WO 2004/018467, WO 2004/018468, WO 2004/018469, WO 2004/041820, WO 2004/046148, WO 2005/051950, WO 2005/082906, WO 2005/063750, WO 2005/085246, WO 2006/027204, WO 2006/029769, W02007/014886; WO 2004/050658, WO 2004/111051, WO 2005/058901, WO 2005/097798; WO 2006/068163, WO 2007/071738, WO 2008/017670; WO 2007/128721, WO 2007/128724, WO 2007/128761, or WO 2009/121945).
Glucagon-like peptide- 1 receptor agonists can be formulated as therapeutics in the combinational therapy provided herein. In some examples, a GLP-1R agonist can be formulated in combination with additional receptor agonists (i.e., GIP and/or glucagon receptor agonist(s)). A single biagonist or triagonist peptide in the combinatorial rotational therapy herein can act as an agonist for multiple receptors of the peptides, proteins and/or pathways detailed above, or pathways involving hunger, digestion and/or metabolism. For example, a biagonist or triagonist peptide that acts as an agonist of one or more glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors, such as the previously described triagonist peptide can be included in the combination therapy to effect weight loss (Finan et al., Nat Med 21(l):27-36 (2015)).
The peptides and corresponding SEQ ID NOs. are set forth below.
Adiponectin
Adiponectin is a 244-amino-acid-long peptide (SEQ ID NO:2), protein hormone and adipokine, involved in regulating glucose levels as well as fatty acid breakdown (Iglesias, J. J., European Journal of Endocrinology 148 (3): 293- 300). Adiponectin is secreted from adipose tissue into the bloodstream and is abundant in plasma relative to several other hormones. High adiponectin levels correlate with a lower risk of diabetes mellitus type 2 (Li et al., JAMA 302(2): 179- 188 (2009)). Adiponectin plays a role in suppressing metabolic derangements that can result in type 2 diabetes, obesity, atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and an independent risk factor for metabolic syndrome (Ukkola et al., J Mol Med 80 (11): 696-702 (2002); Iglesias, J. J., European Journal of Endocrinology 148 (3): 293-300). Adiponectin in combination with leptin has been shown to completely reverse insulin resistance in mice (Chen et al., Diabetologia 49 (6): 1292-302 (2006)). Adiponectin enhances insulin sensitivity primarily though regulation of fatty acid oxidation and suppression of hepatic glucose production (Li et al., JAMA 302 (2): 179 (2009)). Adiponectin exerts weight reduction effects via the brain, similar to the action of leptin; and adiponectin and leptin can act synergistically (Coppola et al., International Journal of Cardiology. 134 (3): 414-6 (2009)).
Adiponectin self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer, and the trimers continue to selfassociate and form hexamers or dodecamers. Studies showed that the high-molecular weight form of adiponectin is the most biologically active form regarding glucose homeostasis (Kuo et al., IntJ of Obesity 35 (12): 1487-94 (2011)).
Plasma levels of adiponectin are lower in obese subjects than in lean subjects (Ukkola et al., J Mol Med 80 (11): 696-702 (2002)). Adiponectin is secreted into the bloodstream where it accounts for approximately 0.01% of all plasma protein at around 5-10 pg/mL (mg/L). Studies show adiponectin is inversely correlated with body mass index in patient populations (Kuo et al. International Journal of Obesity. 35 (12): 1487-94 (2011)); a meta-analysis did not confirm this association in healthy adults (Renaldi et al., Acta Medica Indonesiana. 41 (1): 20-4 (2009)).
Weight reduction significantly increases circulating adiponectin concentrations (Ukkola et al., J Mol Med 80 (11): 696-702 (2002)). High-molecular- weight adiponectin is associated with a lower risk of diabetes with similar magnitude of association as total adiponectin (Kuo et al. International Journal of Obesity 35 (12): 1487-94 (2011)). Coronary artery disease is positively associated with high molecular weight adiponectin, but not with low molecular weight adiponectin (see Fisman et al., Cardiovascular Diabetology 13 (1): 103 (2014)).
Leptin
Leptin is a 167 amino acid hormone (SEQ ID NO:3) predominantly made by adipose cells and enterocytes in the small intestine that helps to regulate energy balance by inhibiting hunger, which in turn diminishes fat storage in adipocytes. Leptin acts on cell receptors in the arcuate and ventromedial nuclei, as well as other parts of the hypothalamus and dopaminergic neurons of the ventral tegmental area, consequently mediating feeding (Brennan et al. , Nature Clinical Practice. Endocrinology & Metabolism (6): 318-27 (2006); Bouret el al..
Physiological Reviews 95 (1): 47-82 (2015); Elmquist et al., Neuron. 22 (2): 221-32 (1999)). Leptin regulates energy metabolism via activation of arcuate proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus. POMC neurons project to the paraventricular nucleus (PVN), where they induce satiety through activation of the brain MC4 receptors.
In the lateral hypothalamus, leptin inhibits hunger by 1) counteracting the effects of neuropeptide Y, a potent hunger promoter secreted by cells in the gut and in the hypothalamus, and 2) counteracting the effects of anandamide, another potent hunger promoter that binds to the same receptors as THC (Elias et al., Neuron. 23 (4): 775-86 (1999)). In the medial hypothalamus, leptin stimulates satiety by promoting the synthesis of a-MSH, a hunger suppressant (Fekete et al., JNeurosci 20 (4): 1550— 8 (2000)).
Along with insulin (see, e.g., SEQ ID NO: 30) and amylin, leptin signals to the brain in proportion to the amount of fat that is stored in the body. Although leptin reduces appetite as a circulating signal, obese individuals generally exhibit a higher circulating concentration of leptin than normal weight individuals due to their higher percentage body fat (Considine et al., The New England Journal of Medicine 334 (5): 292-295 (1996)). Leptin also reduces appetite in response to feeding, but obese people develop a resistance to leptin, similar to resistance of insulin in type 2 diabetes patients, where elevated levels fail to control hunger and modulate weight in obese people. An important contributor to leptin resistance is changes to leptin receptor signaling, particularly in the arcuate nucleus, however, deficiency of, or major changes to, the leptin receptor itself are not thought to be a major cause. Triglycerides crossing the blood brain barrier (BBB) can induce leptin and insulin resistance in the hypothalamus (Fomy-Germano et al., Front in Neurosci 12: 1027 (2019)). Triglycerides can also impair leptin transport across the BBB. id. Leptin interacts with amylin, a hormone involved in gastric emptying and creating a feeling of fullness. When leptin and amylin were given to obese, leptin- resistant rats, sustained weight loss occurred. A recombinant leptin analog, Metreleptin (see SEQ ID NO: 34), was approved by the US FDA in 2014 and the European Medication Association (EMA) in 2018 for the treatment of lipodystrophy (Muller et al. , Nat Rev Drug Discov 21 : 201-223 (2022)). While leptin supplementation is effective in individuals with genetic leptin deficiency, leptin supplementation has not been shown to be effective to lower body weight in obese individuals (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). Combinations of small molecule leptin sensitizers withaferin A and celastrol with the hormones extendin 4 (see SEQ ID NO:21), FGF21 (see SEQ ID NO:22), or GLPl/glucagon improve leptin responsiveness in preclinical studies. Several MC4R agonists have been developed and LY2112688, MC4-NN-0453, MK-0493, and AZD2820, although clinical development has ceased (Muller et al., Nat Rev Drug Discov 21(3):201-223 (2021)). Due to its apparent ability to reverse leptin resistance, amylin has been suggested as possible therapy for obesity (Roth etal., PNAS 105 (20): 7257-62 (2008)). Leptin can be a target for developing a therapeutic, in combination with any of the peptides or therapeutics described herein, or known to be involved with metabolic pathways, in accord with a rotational regimen, such as, for example, to mimic satiety.
Oxyntomodulin
Oxyntomodulin is a 37-amino acid peptide (SEQ ID NO:4) hormone produced by the oxyntic (fundic) cells of the oxyntic (fundic) mucosa in the colon.
Oxyntomodulin suppresses appetite; the mechanism by which it does so is not understood. It binds to the GLP-1 receptor and to the glucagon receptor, but it is not known whether the effects of the hormone are mediated through these receptors or through an unidentified receptor.
Oxyntomodulin has been investigated as a blood-glucose regulating agent in connection with diabetes. Oxyntomodulin is a candidate for treating obesity because of its ability to suppress appetite (Shankar et al., Diabetes. 67 (6): 1105-1112 (2018)). In a 4-week study, healthy overweight and obese subjects treated with oxyntomodulin injections had an average weight loss of 2.3±0.4kg compared to those treated with saline who had an average of 0.5±0.5kg (Wynne et aL, Diabetes. 54 (8): 2390-2395 (2005)).
Sermorelin acetate
Sermorelin acetate (GHRH (1-29)) is peptide analog of growth hormone- releasing hormone (GHRH; see SEQ ID NO:29) which is used as a diagnostic agent to assess growth hormone (GH) secretion to diagnose growth hormone deficiency. Sermorelin acetate is a 29-amino acid peptide representing the 1-29 fragment from endogenous human GHRH, thought to be the shortest fully functional fragment of GHRH (Prakash et al., BioDrugs 12(2): 139-57 (1999); Rang, Dale, Ritter & Moore. Pharmacology. Edinburgh, Churchill Livingstone (2005)).
Studies show that longer term treatment (z.e., 5 months) with sermorelin results in increased GH and IGF-1 and increased lean body mass that were not observed with shorter treatment (2-6 weeks) (Corpas et al., J Clin Endocrinol Metab (1992) 75:530-5; Vittone et al., Metabolism (1997) 46:89-96; Khorram et al., J Clin Endocrinol Metab (1997) 82: 1472-9).
Peptide YY (PYY)
Peptide YY (PYY) is a short (36-amino acid) peptide released from cells in the ileum and colon in response to feeding. In the blood, gut, and other elements of periphery, PYY acts to reduce appetite; similarly, when injected directly into the central nervous system, PYY is also anorexigenic, i.e., it reduces appetite (Woods et al., J Clin Endocrinol Metab. 93 (11 Suppl 1): S37-50 (2008)).
The two major forms of peptide YY (PYY) are PYYI-36 and PYY3-36 (SEQ ID NO:37), which have PP fold structural motifs. PYYi 36 is rapidly cleaved by DPP -IV to its major active form, Peptide YY3-36. PYY3-36 is a linear peptide that contains 34 amino acids with structural homology to NPY and pancreatic peptide (Murphy et al., Nature 444 (7121): 854-59 (2006)). PYY3-36 is the most common form of circulating PYY, which binds to the NPY receptor type 2 (Y2R). This receptor is highly expressed in parasympathetic and sympathetic neurons of the periphery as well as in several regions of the CNS, including the limbic and cortical areas and the brainstem (Stadlbauer et al. , Neuroendocrinol. 38: 1-11 (2015)).
Oxyntomodulin and peptide tyrosine-tyrosine (PYY) are released from intestinal enteroendocrine cells following a meal. These circulating hormones are considered to be satiety signals, as they decrease food intake, body weight, and adiposity in rodents (Wynne et al.. Nat Clin Pr act Endocrinol Metab. 2(11), 612-20 (2006)).
While some studies have shown that obese persons have lower circulating levels of PYY postprandially, other studies have reported that they have normal sensitivity to the anorectic effect of PYY3-36. It is unknown whether reduction in PYY sensitivity contributes to obesity rather than the reduction of leptin sensitivity.
The anorectic effect of PYY is a candidate for a future obesity drug (Murphy et al., Nature (2006) 444 (7121): 854-59). Attempts to use PYY directly as a weightloss drug have met with some success (Bartolome et al., Obes Surg. (2002) 12(3):324- 7). PYY decreases food intake and body weight in rodents and humans (Batterham et al., Nature (2002) 418: 650-654; Batterham et al., N. Engl. J. Med. (2003) 349:941- 948). Researchers noted the caloric intake during a buffet lunch offered two hours after the infusion of PYY was decreased by 30% in obese subjects (P<0.001) and 31% in lean subjects (P<0.001) (Batterham et al., The New England Journal of Medicine (2003) 349 (10): 941 -8). Several PYY analogues have been developed and have entered clinical trials for weight loss treatment including NN9748, NNC0165-1875, and NNC0165-1875 + semaglutide (Poulsen, et al., Pharm. Res. (2021) 8: 1369— 1385).
Protein consumption boosts PYY levels, so some benefit was observed in experimental subjects in reducing hunger and promoting weight loss (Batterham et al., Cell Metabolism. 4 (3): 223-233 (2006)). This can partially explain the weightloss experienced with high-protein diets, but the high thermic effect of protein appears to be the leading cause.
Obese patients undergoing gastric bypass showed marked metabolic adaptations, resulting in frequent diabetes remission 1 year later. When the confounding of calorie restriction is factored out, P-cell function improves rapidly, very possibly under the influence of enhanced GLP-1 responsiveness. Insulin sensitivity improves in proportion to weight loss, with a possible involvement of PYY (Nannipieri et al., J. Clin. Endocrinol. Metab. 98 (11): 4391-9 (2013)).
Amylin The Amylin peptide hormone is co-secreted with insulin from the pancreatic P-cells at a ratio of approximately 100: 1 (insulimamylin) into the blood circulation and is cleared by peptidases in the kidney, so it does not occur in the urine (Higham et al., Eur. J. Biochem. 267 (16): 4998-5004 (2000)). Amylin functions as part of the endocrine pancreas and plays a role in glycemic regulation by slowing gastric emptying and promoting satiety, thereby preventing post-prandial spikes in blood glucose levels.
Amylin's metabolic function is well-characterized as an inhibitor of the appearance of nutrients [especially glucose] in the plasma (Pittner et al., J. Cell. Biochem 55 Suppl: 19-28 (1994)). The overall effect is to slow the rate of appearance (Ra) of glucose in the blood after eating; this is accomplished via coordinate slowing down gastric emptying, inhibition of digestive secretion [gastric acid, pancreatic enzymes, and bile ejection], and a resulting reduction in food intake. Appearance of new glucose in the blood is reduced by inhibiting secretion of the gluconeogenic hormone glucagon. These actions, which are mostly carried out via a glucosesensitive part of the brain stem, the area postrema, can be over-ridden during hypoglycemia. They collectively reduce the total insulin demand (Ratner etal., Diabetic Medicine 21 (11): 1204-12 (2004)).
The clinical application of native amylin in treating obesity is hampered by aggregation and pancreatic islet death (Ling et al., Curr. Protein Pept. Sci. 20, 944- 957 (2019)). Several Amylin peptide analogs have been developed and include Pramlintide (see SEQ ID NO:38), Cagrilintide (SEQ ID NO:43), and ZP 8396. A 2008 study reported a synergistic effect of a human leptin (metrelepin) and an amylin analog (pramlintide) for obesity treatment in diet-induced obese rats and human subjects by restoring hypothalamic sensitivity to leptin (Roth et al., PNAS 105(20):7257-62 (2008)). Pramlintide is approved for patients with Type 1 and Type 2 diabetes in combination with insulin, metformin, or sulfonylurea. Cagrilintide is a long-acting amylin analogue amenable for once-weekly dosing. Dual-acting amylin and calcitonin receptor agonists can induce weight loss in pre-clinical models of obesity (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). Dual agonists include davalintide (AC2307; SEQ ID NO:27), KBP-088 (see SEQ ID NO:31), KBP- 089 (see SEQ ID NO:32), and KBP-042 (SEQ ID NO:42). Tesamorelin
Tesamorelin is a 44 amino acid synthetic form of growth-hormone-releasing hormone (GHRH) which is used in the treatment of HIV-associated lipodystrophy, approved initially in 2010. It is produced and developed by Theratechnologies, Inc. of Canada. Tesamorelin is sold under the trademark Egrifta® medication.
Released GH binds with receptors present on various body organs and regulates body composition; the regulation is primarily due to the combination of anabolic and lipolytic mechanisms. The main mechanisms by which tesamorelin reduces body fat mass are lipolysis followed by reduction in triglyceride levels (Benedini et al., BioDrugs. 22 (2): 101-12 (2008)).
Pancreatic polypeptide
Pancreatic polypeptide (PP) is a 36 amino acids long peptide (human sequence of mature PP set forth in SEQ ID NO:40 (APLEPVYPGD NATPEQMAQY AADLRRYINM LTRPRY) that regulates pancreatic secretion activities by both endocrine and exocrine tissues. PP is synthesized as a 95 aa polypeptide precursor in the pancreatic islets of Langerhans. PP also affects hepatic glycogen levels and gastrointestinal secretions. PP secretion in humans increases after a protein meal, fasting, exercise, and acute hypoglycemia, and decreases after increased food intake and in patients with anorexia nervosa. On fasting, pancreatic polypeptide concentration is 80 pg/mL; and after meal consumption the levels rise to 640 to 800 pg/mL.
Glucose and fats also induce PP and increase the PP level; however, upon parenteral introduction, the hormone levels do not change. Peripheral administration of PP has been shown to decrease food intake in rodents (Batterham et al., The Journal of Clinical Endocrinology and Metabolism. 88 (8): 3989-92 (2003)). PP inhibits pancreatic secretion of fluid, bicarbonate, and digestive enzymes; stimulates gastric acid secretion; is the antagonist of cholecystokinin; and opposes pancreatic secretion stimulated by cholecystokinin (Washabau, Canine and Feline Gastroenterology. Philadelphia, PA, Saunders (2013)).
Gastric Inhibitory Polypeptide
Gastric Inhibitory Polypeptide (GIP2, herein) is an endogenous 42-amino acid peptide synthesized by K cells, which are found in the mucosa of the duodenum and the jejunum of the gastrointestinal tract (Costanzo, Physiology. Philadelphia, PA, SaundersZElsevier (2014)). This endogenous hormone known to be upregulated post Rou-en-Y gastric bypass. GIP is thought to induce insulin secretion, which is stimulated primarily by hyperosmolarity of glucose in the duodenum which has led to referencing GIP as glucose-dependent insulinotropic peptide, while retaining the acronym ”GIP” (Thorens, Diabete & Metabolisme. 21 (5): 311 8 (2014)). In addition to its role as an incretin, GIP is known to inhibit apoptosis of the pancreatic beta cells and promote their proliferation.
Enterostatin
Enterostatin is a peptide previously shown to selectively reduce fat intake, lower cholesterol, reduce angiogenesis, and regulate analgesia. Enterostatin is produced in the gastric mucosa and epithelium of the small intestine, is absorbed from the digestive tract, and functions as a strong anorectic peptide to selectively decrease fat consumption in rodents (D.L. Nelson, D.R. Gehlert, in Comprehensive Medicinal Chemistry II, 2007). Enterostatin interacts with a variety of hormone systems and receptors (Charlotte Erlanson-Albertsson, in Handbook of Biologically Active Peptides (Second Edition), 2013). Enterostatin knock-out mice showed significantly increased serum cholesterol compared to wild-type controls and administration of enterostatin and its peptide fragment DPR reduce serum cholesterol after oral administration in mice (Miller et al., Am J Physiol Endocrinol Metab. 297(4):E856-65 (2009); Takenaka et al., Biosci Biotechol Biochem. 67(7): 1620-22 (2003)).
Three enterostatin protein sequences have been identified in rats and humans, designated VPDPR (rat and human) (see SEQ ID NO:20), APGPR (human; SEQ ID NO: 18), and VPGPR (rat; SEQ ID NO: 19), and have been shown to reduce food intake after chronic ingestion of a high-fat diet in preclinical animal models. In a phase II randomized placebo-controlled trail of 18 obese patients, intravenous enterostatin administration did not impact feelings of hunger, satiety, or food preference.
Ghrelin
Ghrelin (SEQ ID NO:23) is a stomach-derived peptide hormone that signals to the hypothalamus to stimulate homeostatic food intake and hunger; ghrelin levels increase before a meal and when a person is hungry, and levels decrease after a meal. Daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. Strategies to target Ghrelin include suppressing levels of the hormone or antagonizing its receptor, growth hormone secretagogue receptor (GHSR) (Muller et al., Nat Rev Drug Discov 21 : 201-223 (2022)). GHSR antagonists include antimicrobial peptide 2 (LEAP2). Approaches to decrease circulating Ghrelin include the peptide vaccine CYT009-GhrQb, the peptide-binding compounds Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15).
Following sleeve gastrectomy, ghrelin levels decreased and remained low for at least 5 years following surgery (Bohdjalian et al., Obes Surg 20:535-540 (2010)). Fasting ghrelin also decreased after laparoscopic sleeve gastrectomy and postsurgical circulating ghrelin decreased following Roux-en-Y gastric bypass, compared to both obese and normal weight control subjects.
The role of ghrelin in satiety and hunger and the changes in ghrelin regulation and expression following weight loss surgery and between normal and overweight individuals indicate the ghrelin pathway can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
Cholecystokinin (CCK)
Multiple isoforms of CCK are released after food consumption. CCK mediates satiety by acting on CCK receptors distributed throughout the central nervous system. In response to meal initiation, plasma CCK levels have been reported to rise within 15 minutes (Liddle et al., J Clin Invest. 75(4): 1144-1152 (1985)). CCK also has stimulatory effects on the vagus nerve, which can be inhibited by capsaicin (Holzer et al. , American Journal of Physiology. Gastrointestinal and Liver Physiology 275 ( 1 ) : G8-G13 (1998)). The stimulatory effects of CCK oppose those of ghrelin, which has been shown to inhibit the vagus nerve (Kobelt et al., American Journal of Physiology 288 (3): R751-8 (2005)).
CCK was the first gut hormone reported to affect appetite and has been shown to dose-dependently reduce food intake in both rats and humans (Gibbs et al., Nature. 245: 323-325 (1973); Lieverse et al., Gut. 36(2): 176-179 (1995)). The effects of CCK vary between individuals, and are demonstrated in animal studies. For example, in rats, CCK administration significantly reduces hunger in adult males, but is less effective in younger subjects, and less effective in females. The hunger-suppressive effects of CCK also are reduced in obese rats (Fink et al.. Experimental Brain Research 123 (1-2): 77-83 (1998)).
Within the GI tract, CCK is predominantly synthesized and released from the duodenum and jejunum, where its local regulatory effects include stimulation of gallbladder contraction and inhibition of gastric emptying (Buffa et al.. Gastroenterology. 70(4): 528-532 (1967; Dufresne et al., Physiol Rev. 86(3): 805- 847 (2006)). The mechanism for hunger suppression by CCK is thought to be related to a decrease in the rate of gastric emptying (Shillabeer et al., he American Journal of Physiology 252(2): R353-R360 (1987)).
The role of CCK in appetite and food intake and hunger suppression by CCK indicate CCK can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
Vasoactive intestinal peptide
Vasoactive intestinal peptide (VIP) is an endogenous peptide hormone that is 28 amino acid residues. In the digestive system, VIP was shown to induce smooth muscle relaxation (lower esophageal sphincter, stomach, gallbladder), stimulate secretion of water into pancreatic juice and bile, and cause inhibition of gastric acid secretion and absorption from the intestinal lumen (Bowen et al.. “Vasoactive Intestinal Peptide,” Pathophysiology of the Endocrine System: Gastrointestinal Hormones. Colorado State University). VIP stimulates intestinal secretion of water and electrolytes and relaxation of enteric smooth muscle, dilating peripheral blood vessels, stimulating pancreatic bicarbonate secretion, and inhibiting gastrin-stimulated gastric acid secretion (“Vasoactive intestinal polypeptide”. General Practice Notebook. Retrieved 2009-02-06). These effects work together to increase motility (Bergman et al., Atlas of Microscopic Anatomy: Section 6 - Nervous Tissue. URL :. anatomyatlases, org) .
VIP can be a target for weight loss and for inclusion in the rotational combinatorial therapy described herein.
Glicentin
Glicentin is a 69 amino acid peptide secreted after the processing of proglucagon along with the following peptides listed below:
• Pre Proprotein (Proglucagon Preproprotein) (SEQ ID NO: 13) • Signal peptide (1-20; SEQ ID NO:39) - removed from preproglucagon to form proglucagon
• Glicentin (21-89) (Glicentin 21-29 is set forth in SEQ ID NO:24)
• Glicentin-related pancreatic polypeptide (GRPP, 21-50; set forth in SEQ ID NO:25)
• Oxyntomodulin (OXY or OXM, 53-89; set forth in SEQ ID NO:4)
• Glucagon (53-81; set forth in SEQ ID NO:27)
• Glucagon-like peptide 1 (GLP-1, 92-128 set forth in SEQ ID NO: 1) - first seven residues further cleaved
• Glucagon-like peptide 2 (GLP-2, 146-178; set forth in SEQ ID NO:28) Thus, Glicentin contains the entire sequence of glucagon and glicentin-related pancreatic polypeptide. There is no known glicentin receptor, so the mechanism of action is not well characterized. However, the concentration of glicentin in plasma is altered in patients experiencing diabetes and obesity, and following foregut surgery. Glucagon regulates systems metabolism including regulating acute and chronic thermogenic affects. (Kleinert et al., IntJMol Sci. 20(21): 5407 (2019))
GDF15
Macrophage inhibitory cytokine 1 (MIC1; also known as GDF15; SEQ ID NO:33) reduces body weight through appetite suppression. Exogenous administration of recombinant GDF15 or GDF15 analogues decreases body weight in diet-induced obese mice and non-human primates (Mullican, S. E. et al., Nat. Med. 23, 1150-1157 (2017)). GDF15 agents in phase 1 clinical studies include LA-GDF15 and the GDF15 agonists LY-3463251 and JNJ-9090/CIN-109.
Summary
Any of the peptides listed above, and other peptides and therapeutics that are known or thought to impact (i.e., accelerate or initiate) weight loss or that are associated with a pathway involving hunger or digestion or metabolism can be administered in combinations and/or in accord with the rotational protocol set forth herein. In some examples the peptides can be a synthesized peptide that can act as an agonist (or antagonist depending upon the pathway) for a receptor or receptors of any of the peptides and proteins and pathways detailed above, or pathways involving hunger, digestion and/or metabolism. In some examples, a single peptide in the combination and combinatorial rotational therapies provided herein can act as an agonist for multiple receptors of the peptides, proteins and/or pathways detailed above, or pathways involving hunger, digestion and/or metabolism. A combination of any of peptides and/or other therapeutics administered in combination and/or in accord with a rotational regimen, increases the efficacy of weight loss compared to administration of monotherapy or administration of a combination without the rotational component. a. Therapeutic Combinations and Regimens
This section details how protocols provided herein have been developed and successfully employed for obesity, as an exemplary disease, disorder, or condition, and the working examples include protocols for treatment of overweight and obesity and other diseases, disorders, and conditions.
The protocols described herein and below are based on research that shows changes in hormonal secretions after bariatric surgery (see e.g., lonut et cd.. (2015) Obesity 21(6): 1093-1103). The gut-brain axis is a major component of appetite regulation. The gut hormones have either anorexic or orexigenic actions on food intake and it is now evident that these gut hormone secretions are altered following bariatric surgery. Some of the key peptides for administering in the combinatorial and/or rotational combinatorial therapy described herein, and their basal hormone levels and levels after bariatric surgery as shown in previous studies, are listed in Table 10, below (see also Tak et al., Curr Obesity Reports (2021) 10: 14-30), with reference to gastro-neuropathology and with their physiologic influences on the gut-brain. Mechanisms of weight loss drugs, such as AGRP, agouti-related peptide (SEQ ID NO: 14); ARC, arcuate nucleus; CART, cocaine- and amphetamine regulated transcript (SEQ ID NO: 16); DAT, dopamine active transporter; DIR, dopamine 1- class receptor; D2R, dopamine 2-class receptor; GABA, gamma aminobutyric acid; GAB AAR, y-aminobutyric acid type A receptor; GLP-1R, glucagon-like peptide-1 receptor (SEQ ID NO:26); MC3R, melanocortin-3 receptor; MC4R, melanocortin-4 receptor; MOPR, p-opioid receptor; NAc, nucleus accumbens; NPY, neuropeptide Y (SEQ ID NO:35); POMC, proopiomelanocortin; VTA, ventral tegmental area; Y1R, neuropeptide Y receptor type 1; GLP-1R, glucagon-like peptide-1 receptor. Table 10: Exemplary Peptides for Administration for Weight Loss
The data presented herein demonstrate an integrated model for the roles of these gut/adipose/brain peptides and others to regulate appetite in a synchronous fashion, similar to the suppression demonstrated in surgical gastric bypass patients. Due to downregulation of receptors and tolerance that develops almost inevitably with a pharmacotherapeutic agent, regimens provided herein not only include combinations of therapeutics, but also can include a rotation of differing therapeutics, such as, for example, therapeutic peptides, such as rotating a peptide or combinations of peptides every 3 months. In other examples, the combination therapy can be rotated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 weeks or longer, such as every 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 months, or more. In some examples, the regimen can include rotation of different peptides every three months. This rotation will continue to drive weight loss as different receptors even among peptides within the same families will be activated. b. Combination Drug Therapy to Mimic Gastric Bypass
Various hormone levels increase following bariatric surgery, are thought to contribute to the weight loss following bariatric surgery, and the modified levels persist and are long lasting. The combination therapy described herein can be designed to mimic the hormonal and other signaling changes that occur after bariatric surgery, such as Roux-en-Y (Ochner etal., Int JObes 35(2): 153-166 (2011)). The table below includes targets/pathways for targeting for weight loss, exemplary medications for targeting (z.e., activating or inhibiting) the pathways, the function of the administered medication, and exemplary references detailing the target pathways and/or medications for use for targeting the pathways. Exemplary combinations for mimicking changes that occur following gastric bypass include exemplary medications for 2 or more of the targets listed in the table below:
Additional medications targeting pathways modified after bariatric weight loss surgery also can be added to the combinatorial regimen described herein. Such medications include, for example, those that are not used because they are ineffective as a monotherapy, but as described herein can be effective, or efficacy can be increased compared to monotherapy, when administered in combination with other medications, for example in a combinatorial therapy, or a combinatorial rotational therapy. For example, ghrelin receptor antagonists, medications that decrease circulating ghrelin, anti-ghrelin vaccines (e.g., the peptide vaccine CYT009-GhrQb), ghrelin receptor inverse agonists (GHSR-IA), the peptide-binding compounds (e.g., Nox-Bl 1), and any other medication(s) or therapy that decrease ghrelin or ghrelin- associated activation pathways can be included in the combination therapy herein. In other examples, receptor agonists that mimic the action of peptides that are increased after bariatric surgery can be included (z.e., PYY, CCK, and/or PP receptor agonists). In some examples, drugs for inclusion in the combination therapy herein also can include neuropeptide Y (NPY) antagonists, melanocortin-4 receptor (MC4R) agonists, cannabinoid-1 receptor antagonists, and agonists and antagonists as appropriate for increasing weight loss and/or decreasing the comorbidities of overweight and/or obesity.
I PHARMACEUTICAL PRODUCTION, COMPOSITIONS, AND FORMULATIONS
1. Formulation and Administration of the Combinatorial Therapy
The combinations containing the therapeutics for the combination and/or rotational combinatorial therapy described herein can be formulated as pharmaceutical compositions provided for administration by a desired route, such as oral, mucosal, intravenous, and others. Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans, and also, for agricultural applications, for plants. Typically, compounds are formulated into pharmaceutical compositions using techniques and procedures well- known in the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).
An advantage of oral drug delivery compared with injection or nasal spray administration are greater acceptability, ease of administration and convenience for patients. With the protocol described herein, oral delivery of a combination of medications (i.e., therapeutic peptides) would increase compliance over frequent subcutaneous injections.
Oral delivery systems can pose a challenge due to proteolytic cleavage in the digestive tract, limited intestinal uptake of the intact molecule, and absorption of peptides that can be hindered by their physical characteristics, including the polarity and size of the dosage form, and their susceptibility to aggregation or degradation by the local pH in the tract. As described herein, most delivery vehicles cannot be administered orally, but some can be so-administered. Such vehicles can be selected if oral delivery is desired.
Various strategies have been adopted to increase bioavailability of proteins and peptides. Different approaches can be used to enhance systemic exposure of peptides following oral delivery of the combination therapy described herein. In some examples, one or more of the following mechanisms can be utilized to enhance delivery of the combination of therapeutics described: 1) permeation enhancers with added glycosylation, PEGylation or lipidation to increase uptake across cell membranes; 2) enzyme inhibitors such as, for example, carnitine esters, soybean trypsin inhibitors, deoxycholic acids and organic acids to prevent degradation in the gut; 3) modulation of protein to prevent degradation, such as, for example, by adding stabilizing side chains to reduce degradation; 4) additional of particulate systems, as described herein, including polymeric micro and nanoparticles, liposomes, microemulsification or polymeric micelles; 5) addition of multifunctional polymers, to include, for example, Poly (alky cyanacryl ate) chitosan to assist with mucosal adhesion and bypass tight junctions; and 6) modification of ligand-specific binding and uptake, to include binding to B12, biotin, lecithin, and folate.
The therapeutic molecules in the combinations can be administered in forms that increase half-life. For example, the therapeutic molecules in the combinations can be provided as part of a liposome or multicellular laminar vesicle or other such delivery vehicle.
The therapeutic molecule provided in the combinations herein can be modified by a polymer prior to administration. In some examples, the polymer is a polyalkylene glycol, dextran, pullulan or cellulose. Polyalkylene glycol polymers, which can modify the therapeutic molecule include polyethylene glycol (PEG) and methoxypolyethylene glycol (mPEG). In examples where the therapeutic molecule is modified by PEG, the PEG can by branched or linear. In some embodiments, the polymer can be produced by reaction with methoxy -poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (5 kDa); methoxy-poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (20 kDa); methoxy-poly(ethylene glycol)- succinimidyl butanoate (mPEG-SBA) (30 kDa); methoxy -poly(ethylene glycol)- succinimidyl a-methylbutanoate (mPEG-SMB) (20 kDa); methoxy-poly(ethylene glycol)-succinimidyl a-methylbutanoate (mPEG-SMB) (30 kDa); methoxypolyethylene glycol)-butyraldehyde (mPEG-butyraldehyde) (30 kDa), m ethoxy - poly(ethylene glycol)-succinimidyl propionate (mPEG-SPA) (20 kDa); methoxy- poly(ethylene glycol)-succinimidyl propionate (mPEG-SPA) (30 kDa); (methoxy- poly(ethylene glycol)) 2-N-hydroxysuccinimide ester (mPEG2-NHS) (10 kDa branched); (methoxy-poly(ethylene glycol)) 2-N-hydroxysuccinimide ester (mPEG2- NHS) (20 kDa branched); (methoxy -poly(ethylene glycol)) 2-N-hydroxysuccinimide ester (mPEG2-NHS) (40 kDa branched); (methoxy -poly(ethylene glycol)) 2-N- hydroxysuccinimide ester (mPEG2-NHS) (60 kDa branched); biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) (5 kDa biotinylated); poly(ethylene glycol)-p-nitrophenyl carbonate (PEG-p-nitrophenyl-carbonate) (30 kDa); or poly(ethylene glycol)-propionaldehyde (PEG-propionaldehyde) (30 kDa).
Any of the above approaches can be used to formulate the combination(s) of therapeutics described herein. The combinations can be used for therapeutic, prophylactic, cosmetic, and/or diagnostic applications. The combinations containing the therapeutics for the rotational combinatorial therapy described herein can be formulated with a pharmaceutical acceptable carrier or diluent. Generally, such pharmaceutical combinations of compositions include components that do not significantly impair the biological properties or other properties of the cargo.
Each component in the combination is pharmaceutically and physiologically acceptable so that it is compatible with the other ingredients and not injurious to the subject to whom it is to be administered.
The formulations can be provided in unit dosage form and can be prepared by methods well-known in the art of pharmacy, including but not limited to, tablets, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, oil water emulsions, liquid solutions or suspensions (e.g., including injectable, ingestible and topical formulations (e.g., eye drops, gels, pastes, creams, or ointments)), aerosols (e.g., nasal sprays, and inhalers), liposomes, suppositories, pessaries, injectable and infusible solution and sustained release forms. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. Each unit dose contains a predetermined quantity of therapeutically active compound or compounds sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dose forms include ampoules. When administered systemically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art. Methods for preparing parenterally administrable compositions are well-known or will be apparent to those skilled in the art and are described in more detail in, e.g., “ Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences)”, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
Pharmaceutical combinations provided herein can be in various forms, e.g., in solid, semi-solid, liquid, powder, aqueous, and lyophilized form. Examples of suitable pharmaceutical carriers are known in the art and include but are not limited to water, buffering agents, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, and powders, among others. Pharmaceutical compositions provided herein can contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and miscellaneous ingredients such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, and starch, among others (see, generally, Alfonso R. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body.
The therapeutics for the rotational combinatorial therapy described herein can be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulation and administration of the compounds are known to one of skill in the art (see e.g., “Remington's Pharmaceutical Sciences.'' Mack Publishing Co., Easton, Pa.). This therapeutic composition can be administered intravenously or through the nose or lung, such as a liquid or powder aerosol (lyophilized). The composition also can be administered parenterally or subcutaneously as desired. When administered systematically, the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
2. Dosage Forms
Therapeutic formulations can be administered in many conventional dosage formulations. Dosage formulations of therapeutics for the rotational combinatorial therapy described herein can be prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and can include buffers such as Tris HC1, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, di saccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN (polysorbates), Pluronic, polyethylene glycol, and others.
When used for in vivo administration (z.e., to patients), the formulation should be sterile and can be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. For example, a therapeutic peptide for a rotational combination therapy described herein can be provided as lyophilized powder that is reconstituted with a suitable solution to generate a single dose solution for injection. The therapeutics for the rotational combinatorial therapy described herein can be stored in lyophilized form or in solution; they can be frozen or refrigerated. In some embodiments, the lyophilized powder can contain the combination of therapeutics, such as a combination of therapeutic peptides, and additional components, such as salts, such that reconstitution with sterile distilled water results in a combination of therapeutics in a buffered or saline solution.
Unit dose forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials and syringes. Hence, multiple dose form is a multiple of unit doses that are not segregated in packaging.
The therapeutics for the rotational combinatorial therapy described herein can be provided at a concentration in the composition that is approved by regulatory agencies. For example, for FDA approved pharmaceuticals, the pharmaceuticals are formulated and provided at a concentration for which the pharmaceuticals are approved (ie., as monotherapy or dual therapy). In some examples, the pharmaceutical composition contains multiple therapeutics in a combination and multiple different therapeutics for the rotational combinatorial therapy described herein. In some examples, the combination therapy described herein includes one or more additional agents, such as treatment of adverse side effects, or other therapeutic, for combination therapy.
3. Dosage and Administration
Pharmaceutical compositions suitable for use include compositions wherein the therapeutics for the rotational combinatorial therapy described herein are contained in an amount effective to achieve their intended purpose. For example, previously FDA approved medication can be in an amount or dosage as previously approved or recommended by the FDA or any other regulatory agency. In other examples, the FDA approved medication used in a combination described herein can be provided in a lower amount or dosage than approved or recommended by the FDA or other regulatory agency. In some examples, multiple medications are provided in the combination in an amount or dosage that is approved or recommended by FDA. In other examples, multiple medications are provided in the combination in an amount or dosage that is less than the amount or dosage approved or recommended by FDA. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Therapeutically effective dosages can be determined by using in vitro and in vivo methods, and/or by a skilled person.
Dosages generally start at FDA approved or recommended dosage. The lowest effective dosage can be indicated in order to decrease side effects. Dosages can be adjusted throughout the therapeutic period in accord with the regimen herein and also based on other factors. For example, dosage can be modified based on the particular subject’s response and/or progress (z.e., weight loss), tolerability, volume of distribution, subject weight and/or body size, and at the discretion of the treating physician. Metabolic parameters, cardiac markers for cardiac patients (z.e., CRP, ASR), inflammatory markers (z.e., myeloperoxidase (MPO)), renal clearance also can be assessed when determining dosage; dosages to decrease inflammation, decrease cardiac risk and that are renal protective would be preferred so long as the therapeutic efficacy is maintained.
Any of the therapeutics for the rotational combinatorial therapy or combinations described herein can be formulated for single dosage or multiple dosage administration for use in the rotational therapy provided herein; generally, the therapeutics are formulated for multiple dosage. In some examples, an approved (z.e., FDA approved) therapeutic that is approved for single dosage administration is formulated for single dosage administration in the combinatorial therapy provided herein. In other examples, an approved (z.e., FDA approved) therapeutic that is approved for multiple dosage administration is formulated for multiple dosage administration in the combinatorial therapy provided herein. For example, a therapeutic that is approved for daily administration can be administered daily in the rotational combinatorial regimen described herein. In some examples of multiple dosage administration, a therapeutic can be included in a first combination and rotated into the regimen as part of a later combination (z.e., fourth or fifth combination). In other examples, a therapeutic is provided in a combination and is not rotated into the regimen again; such as a therapeutic that is provided in a first combination only, and is not included in a later combination in the regimen.
The range of doses of any of the therapeutics in the provided combinations can be formulated per kg of body weight of the subject or patient. The dose can be administered a single time during each rotation, or can be administered a multiple times. Appropriate dose amount can be determined by one of skill in the art, based on the regimen of administration. Total dose over a specific period of time can also be selected by one of skill in the art. In a rotational combinatorial therapy herein, the dose can be increased over time, during the regimen. In some examples the dose can be increased or decreased during the rotation, compared to the previously administered dose.
The dose range for each of the individual therapeutics in the combinations provided herein, can be adjusted by plasma monitoring or by monitoring of symptoms or by monitoring of adverse side effects. The dose of administration can be such that the subject will maintain a plasma level to effect amelioration of symptoms of the disease, disorder, or condition for which treatment is administered. The dose of an individual therapeutic can vary depending on the other therapeutics in the combination, and can vary depending on the doses of the therapeutics in the other combinations in the rotation.
The therapeutics and combinations are included in amounts sufficient to exert a therapeutically useful effect in the absence of undesirable side effects, or with minimal or decreased side effects on the patient treated. A therapeutically effective concentration of a therapeutic(s) for treatment of any condition with a previously characterized mechanism of action or pathway can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art and then extrapolated therefrom for dosages for humans.
The precise amount or dose of the therapeutic agent administered depends on the particular agent, the route of administration, the other agents in the combination, the other agents previously administered in the rotational regimen, the amounts (z.e., dosages) of the other agents previously administered in the rotational regimen, and other considerations, such as the severity of the disease and the weight and general state of the subject. Local administration of the therapeutic agent will typically require a smaller dosage than any mode of systemic administration, although the local concentration of the therapeutic agent can, in some cases, be higher following local administration than can be achieved with safety upon systemic administration. If necessary, a particular dosage can be empirically determined or extrapolated. In some examples, the dosage of an approved (z.e., FDA approved) medication is the approved dosage or amount, or a standard dosage or amount, or a dosage or amount provided in applicable clinical guidelines (z.e., clinical guideline from the American Society for Radiation Oncology (ASTRO) provides guidance on the use of radiation therapy). In some examples, the dosage of a therapeutic in a combination is less than the dosage or amount approved by a regulatory agency. For example, the dosages of a therapeutic (z.e., medication) in a combination can be decreased compared to the dosage of the therapeutic when administered as a monotherapy.
The amount of any therapeutic in the combinations provided herein to be administered for the treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify dosage ranges for administration. For drugs that do not have established dosages (z.e., FDA approved dosages) animal models can be used to establish an effective dosage. One of skill in the art can determine the human equivalent dose (HED), based body weight for humans. Appropriate HED can be calculated using body weight-based conversion (Reagan-Shaw et al. (2008) The FASEB Journal 22(3):659-661). The precise dosage, which can be determined empirically or based on previous use of the therapeutic (z.e., the standard dosage), can depend on the particular agent, the route of administration, the type of disease to be treated, the particular disease or condition, the particular formulation, the seriousness of the disease or condition, and other factors within the level of a skilled artisan. In some embodiments, the combinations administered can contain therapeutic peptides that mimic the peptides released or increased following bariatric weight loss surgery provided herein, such as, for example, gastrointestinal hormones, PYY and/or GLP-1. In examples, the therapeutic peptides are administered in a dosage that effects levels (z.e., circulating levels) of the peptides similar to the levels following bariatric weight loss surgery. In some examples, the dosage of an individual therapeutic in the combination is the dosage approved by a regulatory agency for administering an approved therapeutic as a monotherapy.
It is understood that the precise dosage and duration of treatment is a function of the disease being treated and the dosage and duration of treatment can follow a previously established schedule for administration of an approved therapeutic (z.e., drug). Therapeutic dosage and duration of treatment also can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. One of skill in the art can, in a clinical setting, and in accord with the rotational methods and combinations described herein, determine the dosage of individual therapeutics in the combinations provided herein for administration. Dosage and duration of treatment also can be modified considering the other therapeutics in the combination, and in the rotational therapy. Concentrations and dosage values also can vary with the severity of the condition to be alleviated. The dosages can depend on the clinical response and side effect profile of an individual subject, such as a human or an animal.
For any particular subject, specific predetermined dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the combination therapy. The concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of combinations containing them. Generally, dosages and dosage regimens for the combination therapy are chosen to limit toxicity and/or adverse side effects. For example, the therapeutic can initially be administered at a low dosage and increased gradually according to patient's response. In another example, the therapeutic can initially be administered at the standard dosage and can be increased gradually according to the patient’s response.
The combinations can be administered hourly, daily, bi-weekly, weekly, monthly, once, twice daily, three times a day, four times a day, five times a day, or more. In some examples, different therapeutics in a combination are administered with the same frequency. For example, three therapeutics in a first combination are administered once daily. In another example, four therapeutics in a first combination are administered twice daily. The frequency of administration can vary as long as all of the therapeutics are administered with the same frequency (z.e., at the same time). In other examples, therapeutics in a combination are administered on distinct schedules (z.e., with different frequency). For example, one therapeutic in a first combination is administered twice daily, and a second therapeutic in the first combination is administered once daily, and a third therapeutic in the first combination is administered bi-weekly. Generally, the therapeutics are administered in accord with their approved administration monotherapy timing. For example, therapeutics that are administered daily as a monotherapy are administered daily in the combination regimen provided herein. In other examples, therapeutics that are administered weekly as a monotherapy are administered weekly in the combination regimen provided herein. In other examples, therapeutics that are administered biweekly as a monotherapy are administered bi-weekly in the combination regimen provided herein.
4. Dosage and Administration for Treating Obesity and Overweight
The delivery vehicles, such as the liposomes, provided herein can be formulated for administration by any suitable route, including oral and injection, such as subcutaneous injection. For example, liposomes displaying combinations of peptides as exemplified, can be formulated for subcutaneous administration, particularly for self-administration. Numerous injectors, autoinjectors, and high speed autoinjectors are known in the art see, e.g., U.S. Patent Nos. 618,396, 7,731,686, 8,021,335, 8,647,299, 8,945,067, 9,333,304, 9,408,973, 9,421,337, 10,493,212, 10,695,492, 10,716,901, 11,058,820, 11,167,087, 11,285,266, 11,648,355, 11,992,663 and 12,005,236, 12,208,245, and U.S. patent publication 2024/0207375. An appropriate single dose or multiple dose can be loaded into an injector for administration. In embodiments in which the liposomes display a plurality of peptides (or also contain a small molecule), the dosage amounts of the liposomes can be provided in the injector for use by a subject. A typical volume is 1 mL to 3 mL (or a range therein) or up to about 5 mL, and in some instances, where the small molecule includes about 10 mL. As described herein, the liposomes can be produced that display 1 to 3 peptides, The liposomes can be separately administered or mixed and administered in a single composition, which can be loaded into an injector, such as for subcutaneous administration. Liposomes, such as those exemplified herein, can be administered via injection. Optionally, the small molecules, such as phentermine, can be administered as a separate dosage form, such as a tablet or capsule. They can be administered together, or sequentially or intermittently with the delivery vehicles. Generally, they can be included in or on the delivery vehicles.
In some examples of combination or rotational combinatorial therapy for treating obesity or overweight, the combinations can be formulated using previously approved (ie., approved by the US Food and Drug Administration) dosages. For example, GLP-1 agonists can be formulated at the starting dosage in the combinations that is the starting dosage of the GLP-1 agonist administered as a monotherapy. For example, dulaglutide (Trulicity®) GLP-1 agonist can be formulated in the combination at a starting dosage of 0.75 mg administered once weekly, exenatide (sold under the trademark BYDUREON Bcise®) GLP-1 agonist can be formulated in the combination at a starting dosage of 2 mg administered once weekly, and semaglutide (soled as Ozempic®) GLP-1 agonist can be formulated in the combination at a starting dosage of 0.5 mg administered once weekly. Exemplary medications and their starting dosages are set forth in Table 11, below.
Table 11: Exemplary Medications, Administration and Starting Dosage for
Weight Loss
In some examples of combinations for rotational combinatorial therapy for treating obesity or overweight, the combinations can be formulated and administered in the therapeutic amounts or ranges set forth in Table 12, below:
Table 12: Exemplary Medications, Administration and Dosage Range
Also described herein is a pharmaceutical combination for treating overweight, obesity and/or for weight loss including FDA approved medications. In some examples, one or more of the following is included the combination: 1) a GLP-1 agonist (sold under trademarks Trulicity®, B-cise®, Ozempic®, and Victoza®), 2) Phentermine, 3) Liothyronine, 4) topiramate (sold under the trademark Topamax® carbonic anhydrase inhibitor), 5) Sermorelin, and 6) tirzepatide (sold under the trademark Mounjaro™). In other examples, one or more of the following is administered: one or more GLP-1 agonists, such as, for example Trulicity®, BYDUREON Bcise®, Ozempic®, Victoza® GLP-1 agonist; Phentermine; Liothyronine; topiramate (such as Topamax® carbonic anhydrase inhibitor); Acarbose; Sitagliptin (Januvia® dipeptidyl peptidase-4 (DPP-4) inhibitor); Canagliflozin (Invokana® sodium-glucose co-transporter 2 (SGLT2) inhibitor); Dapagliflozin (Farxiga® SGLT2 inhibitor); tirzepatide; and Sermorelin. In some examples, one or more of the medications in the pharmaceutical combination is discontinued and restarted. For example, one medication (z.e., phentermine) is discontinued for a period (z.e., one month) and another medication (z.e., Liothyronine) replaces the first medication, and after a time period (z.e., 1 month) the first medication is resumed and the replacement medication is stopped. In some examples a medication or medications is started after the first medication(s) is/are administered (the regimen is started), for example 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more after the regimen is started.
Accordingly, the methods and regimens provided herein include a method of treating an obese subject with a rotational combination therapy comprising administering a plurality of combinations of therapeutics to the subject. An effective amount of the therapeutics in the combinations readily is determined by one of skill in the art to effect weight loss in the subject. Doses of the therapeutics in the combinations or the timing of the treatment regimen can be varied or adjusted based on the susceptibility of the patient to the treatment, as determined by one of skill in the art, such as by using the methods provided herein.
When an individual therapeutic provided in a combination herein is coformulated or co-administered with another therapeutic agent or agents in the combination, dosages can be provided as a ratio of the amount of one of the therapeutics in the combination to the amount of the other therapeutic agent(s) administered.
5. Routes of Administration of the Combinations
The therapeutics in the combination therapy and the combinations provided herein can be formulated for any route known to those of skill in the art including, but not limited to, subcutaneous, intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, epidural, vaginal, rectal, local, optic, transdermal administration, or any route of administration. For subcutaneous administration the single dosage is administered in volume that is generally 10 ml or less, such as 1-5 ml, 1-2 ml, 2-10 ml, no more than 4, 5, 6, 7, 8, or 9 ml. Formulations suited for such routes are known to one of skill in the art. Formulations of previously characterized (z.e., FDA approved) therapeutics can be administered in the combinations herein in accord with the previously characterized administration route. Therapeutic compositions also can be administered concomitantly with other therapeutics in the combination. Therapeutic compositions also can be administered with other biologically active agents including therapeutics in the combination or other biologically active agents, either sequentially, intermittently or in the same composition. The combination therapy can be administered by any suitable route, and for any use for which the combination of therapeutics are used, including treatment of obesity or overweight.
Pharmaceutical compositions can be administered by controlled release formulations and/or delivery devices (see, e.g., in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,660; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,556; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). Various delivery systems are known and can be used to administer selected compositions, are contemplated for use herein, and such particles can be easily made.
The route of administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical, rectal, mucosal, and by sustained release systems. The combinations containing the therapeutics for the rotational combinatorial therapy described herein can be administered continuously by infusion or by bolus injection. The skilled physician can administer the combinations containing the therapeutics for the rotational combinatorial therapy described herein in a local or systemic manner. a. Administration of Combinatorial Treatments for Improved Weight Loss
Described herein is an improved pharmacotherapeutic cocktail that mimics bariatric weight loss surgery and includes combining the selected peptides at the right physiologic time (i.e., prior to meals, or early or later in the day) and developing stable and soluble peptides that will be easily administered through subcutaneous injection, or intramuscular injection, or by oral formulation. Each of the peptides described herein form a part of the message which becomes integrated at different levels of the brain and produces an outcome of either feed or do not feed.
Table 13 below sets forth an exemplary protocol for administering a combinatorial therapy, including 6 different combinations, each comprising more than one therapeutics, where the combinations are rotated after 4 months of administration. The therapeutics in the combinations target different pathways, for different effects. Table 13: Exemplary Timing for Peptide Administration
6. Articles of Manufacture and Kits
Pharmaceutical compositions containing the combinations for the rotational therapy described herein can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a disease or condition that can be treated by rotational administration of the particular combinations, such as the diseases and conditions described herein or known in the art, and a label that indicates that the cargo, such as a drug or other therapeutic (z.e., a weight loss medication), is to be used for treating the condition, disease or disorder. The pharmaceutical compositions can be packaged in unit dosage forms containing an amount of the pharmaceutical composition for a single dose or multiple doses. The packaged compositions can contain a lyophilized powder of the pharmaceutical compositions containing the combinations for the rotational therapy which can be reconstituted (e.g., with water or saline) prior to administration in accord with the rotational regimen.
The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252). Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers (e.g., pressurized metered dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. For example, the combinations provided herein can be packaged in accord with the packaging of the individual medications as provided individually, or can be packaged in a different packaging that contains all or some of the components of the combination.
The combinations for rotational therapy can be packaged as kits. Kits optionally can include one or more components such as instructions for use, devices, and additional reagents (e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods. Exemplary kits can include the therapeutic(s) in the combinations for rotational therapy provided herein, and can optionally include instructions for use, a device, such as a syringe or other injector, for administering the combinations for rotational therapy to a subject, and a device for administering an additional therapeutic(s) agent to a subject.
The kit can, optionally, include instructions. Instructions typically include a tangible expression describing the combinations for rotational therapy, and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the combinations for rotational therapy. Instructions also can include guidance for monitoring the subject over the duration of the treatment time. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount, or activity of therapeutic(s) in the combination for rotational therapy, in a subject.
Kits provided herein also can include a device for administering the combinations to a subject. Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a nebulizer, and an inhaler. Typically, the device for administering the compositions is compatible with the desired method of administration.
Devices for injection
Devices for administering the delivery vehicles, such as the liposomes herein are known. In general, the delivery vehicles, such as the liposomes, can be administered by injection, such as subcutaneous or intramuscular injection. The peptide drugs are known and can be delivered by subcutaneous injection, and are commercially available in autoinjectors or other such devices that that deliver predetermined amounts of a product. The product can be provided in the device as a liquid, or the device can contain chambers in which a lyophilized product is separate from a liquid vehicle, such as PBS, for administration. A membrane separating the chambers can be pierced before using and the lyophilized product dissolved in the vehicle. Additionally, the injectors can include multiple chambers, if the drugs are provided, on different liposomes. Appropriate amounts to achieve a desired dosage of each are mixed before injection.
Among the devices that can be used or in which the delivery vehicles, such as liposomes, can be provided are autoinjectors that contain a fixed dosage amount or multiple dosages of the delivery vehicles. Autoinjectors include spring-actuated mechanical devices, which are user administered by pressing a button or pushing against an injection site (push-on-skin), which provides a pre-determined fixed volume from a pre-filled syringe. Autoinjectors can include visual and audible cues, and, in general, permit non-healthcare professionals to administer medications safely and effectively. Autoinjectors for use in the delivery drugs including biologies and small molecules are well known and readily accessible to the public. Examples of approved drugs administered by autoinjectors include, but are not limited to, Epinephrine (Adrenaline) (e.g., EpiPen® autoinjector system), Naloxone (e.g., EZVIO™ naloxone autoinjector), Atropine (and Pralidoxime), Diazepam, Sumatriptan (e.g., Alsuma/StatDose), Abatacept (e.g., ORENCIA® ClickJect™ single-dose prefilled autoinjector), Etanercept (e.g., lyophilized Enbrel® reformulated for the SureClick® autoinjector), Mepolizumab (e.g., NUCALA® autoinjector), and Benralizumab (e.g., Fasenra® autoinjector), and commercially available versions of the weight loss peptides, such as the GLP-1 receptor agonists and others.
Autoinjectors are an be used in the delivery of fixed volumes of injectable fluid, where the injectable fluid contains the delivery vehicles or the delivery vehicles and any suitable excipient. Exemplary autoinjector systems are described in U.S. Pat. Nos. 7,618,396, 7,731,686, 8,021,335, 8,647,299, 8,945,067, 9,333,304, 9,408,973, 9,421,337, 10,493,212, 10,695,492, 10,716,901, 11,058,820, 11,167,087, 11,285,266, 11,648,355, 11,992,663 and 12,005,236, 12,208,245, and U.S. 2024/0207375 U.S. patent publication 2024/0207375 describes high volume autoinjectors for use for subcutaneous administration.
Exemplary routes of administration for auto injection include subcutaneous and intramuscular delivery. Exemplary FDA and EMA approved subcutaneous syringes, prefilled needle safety devices, and prefilled handheld autoinjectors include, for example:
Handheld autoinjectors optimized for doses less than about 2.0 mL (e.g.,
EpiPen®); autoinjectors capable of delivering higher doses, ranging between 2 and 5 mL or more (e.g., YpsoMate® 5.5) also are available. For subcutaneous administration the single dosage is administered in volume that is generally up to 10 mL, or about 10 ml or less, such as 1-5 ml, 1-2 ml, 1-3 ml, 2-10 ml or other such amounts. Auto injection rates range from at least or about 0.003 mL/s to about 1.0 mL/s. For example, a high volume autoinjector can be configured to subcutaneously administer a formulation to a subject at a rate of about 0.05 mL/sec to about 1.0 mL/sec. It is known that the speed at which a high volume of the formulation can be administered to the subject depends on the gauge of the needle used to inject the formulation. The size and injection rate of the autoinjector can be configured to meet the needs of the end-user, see, e.g., U.S. 2024/0207375.
Exemplary commercial handheld autoinjectors at or exceeding 3.0 mL for subcutaneous administration include:
Wearable devices or on-body delivery systems (OBDS) provide for subcutaneous delivery of volumes greater than 3mL. Exemplary devices include SmartDose® 3.5 injector (West Pharma, Exton, PA, USA), CRONO ambulatory infusion pumps (Cane Medical Technology, Rivoli TO, Italy), Infusion Pump (Sensile Medical, Olten, Switzerland), Libertas™ autoinjector (BD, Franklin Lakes, NJ, USA), Lapas® patch pump (Bespak, King’s Lynn, UK), enFuse® on-body platform (Enable Injections, Cincinnati, OH, USA), Wearable injectors (Sonceboz, Sonceboz- Sombeval, Switzerland), Wearable injection devices (Sorrel Medical, Netanya, Israel), DrugDeliverySystems (Weibel, Zug, Switzerland), and YpsoDose® (Ypsomed, Burgdorf, Switzerland). Other high volume autoinjectors are described in U.S. Pat. No. 8,021,335 and U.S. 2024/0207375. Auto injection rates range from at least or about 0.003 mL/s to about 0.1 mL/s, and at least or about 0.1 mL/s to about 0.9 mL/s; higher speed injectors also are available. In general, auto injection volumes range from at least or about 0.1 to about 0.5 mL, at least or about 0.1 to about 1.0 mL, at least or about 0.1 to about 1.5 mL, at least or about 0.1 to about 2.0 mL, at least or about 0.1 to about 2.5 mL, at least or about 0.1 to about 3.0 mL, at least or about 0.1 to about 3.5 mL, at least or about 0.1 to about 4.0 mL, at least or about 0.1 to about 4.5 mL, at least or about 0.1 to about 5.0 mL, at least or about 0.1 to about 5.5 mL, at least or about 0.1 to about 6.0 mL, at least or about 0.1 to about 6.5 mL, at least or about 0.1 to about 7.0 mL, at least or about 0.1 to about 7.5 mL, at least or about 0.1 to about 8.0 mL, at least or about 0.1 to about 8.5 mL, at least or about 0.1 to about 9.0 mL, at least or about 0.1 to about 9.5 mL, and at least or about 0.1 to about 10.0 mL and greater than 10 mL. For purposes herein, the volume is generally about 1-3 mL; higher volumes also are contemplated and autoinjectors for delivering higher volumes are known.
Excipients that facilitate subcutaneous injection can be included in the formulations and pharmaceutical compositions. The delivery vehicles can be coformulated with such excipient, such as with a hyaluronidase, such as a soluble human hyaluronidases, such as the commercially available ENHANZE® soluble human hyaluronidase. Including a hyaluronidase in a formulation, such as those provided herein, reduces dose administration time, dosing frequency, and provides for the delivery of large volumes for rapid subcutaneous injections, such as 5 to 15 mL, and infusions, up to 600 mL. Hence provided are compositions that contain the delivery vehicles and an excipient, such as a hyaluronidase, such as a human soluble hyaluronidase or variant thereof.
J. METHODS OF ASSESSING ACTIVITY, BIOAVAILABILITY AND PHARMACOKINETICS
Pharmacokinetics and tolerability
Pharmacokinetic and tolerability studies can be performed using animal models or can be performed during clinical studies with patients to assess the effect of the combinations provided herein. Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques. In some instances, pharmacokinetic and tolerability studies are performed using healthy animals. In other examples, the studies are performed using animal models of a disease for which therapy with a combination herein is considered, such as animal models of obesity, or animals with metabolic disorders (ie., transgenic animals that model or show characteristics of metabolic disorders). In other examples, animal models of diabetes (e.g., diabetes mellitus type 2), cardiovascular disease (z.e., cardiovascular disease leading to heart attack or stroke), high blood pressure, high blood cholesterol, high triglyceride levels, persisting neurodegenerative disorders, such as Parkinson’s disease, metabolic syndrome, obstructive sleep apnea, cancer, osteoarthritis, depression, and/or nonalcoholic fatty liver disease can be used to assess pharmacokinetics of the combinations herein.
The pharmacokinetic properties and activities of therapeutics in the rotational combinatorial therapy herein can be assessed by direct and indirect methods. For example, the concentration of the therapeutics (z.e., drug), such as, for example, in the blood or serum or other body fluid, can be assessed after therapeutic administration or at a time point following administration, such as, for example, at or about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks or more after administration of the rotational combinatorial therapy. For example, the concentration of the active drug ingredient in the blood can be assessed. In other examples, the equation of bioavailability can be used for assessing the therapeutic effect. In some examples the following formula is used for evaluation: [AUC]test Dstd Ttest / [AUC]std Dtest Tstd, where D is the dose administered, test and std are the test and standard doses of the same drug to determine the relative availability and AUC=area under the curve.
Indirect methods also can be used to assess the pharmacokinetic properties and activities of therapeutics, and the therapeutic effect(s). For example, the rate of urinary excretion, such as cumulative urinary excretion, can be measured or by the pharmacological effects can be assessed. For example, the drug pharmacokinetics can be assessed by evaluating drug absorption, distribution, bioavailability, metabolism and elimination.
A range of doses and different dosing frequency of dosing can be administered in the pharmacokinetic studies to assess the effect of increasing or decreasing concentrations of the therapeutics in the combinations. Pharmacokinetic properties, such as bioavailability, of individual drugs in the combination also can be assessed with co-administration of other therapeutic(s) in the combination. For example, animal models can be administered the therapeutics in a first combination, using one or more routes of administration. Such studies can be performed to assess the effect of co-administration of the plurality of therapeutics (z.e., drugs) in the combination.
Studies to assess safety and tolerability also are known in the art and can be used herein. Following administration of a combination and compositions herein, the development of any adverse reactions can be monitored. Adverse reactions can include, but are not limited to, injection site reactions, such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses. Typically, a range of doses and different dosing frequencies are be administered in the safety and tolerability studies to assess the effect of increasing or decreasing concentrations of the active agent.
K. SEQUENCE SUMMARY
The following table summarizes sequences of peptides and other polypeptides for use in delivery vehicles, compositions, regimens, combinations, and methods provided herein. Precursors and peptides are noted in the tables. Peptides can be displayed or provided in delivery vehicles, such as liposomes. Small molecule drugs and larger polypeptides cand be provided in delivery vehicles, such as liposomes, as described in the Examples, or they can be provided separately for administration, generally for separate administration. For display on the delivery vehicles, such as liposomes, the peptides can be modified to include a K (Lys) near or at the C-terminus for pegylation, such as to link to the liposomes as exemplified and described herein. The position of the K is selected so that, when displayed, the peptide retains activity. The peptides also can be modified to replace other K residues with a conservative amino acids, such as R, so that only the K near the C-terminus (or a locus that does not affect activity) is pegylated.
L. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1
1 g of l-palmitoyl-2-oleoyl phosphatidyl choline and 50 mg dipalmitoyl phosphatidyl ethanolamine is dissolved into 50 ml of chloroform/methanol 3: 1 mixture. 300 mg cholesterol is added. Solvent is removed by rotary evaporator, and then by applying high vacuum using a vacuum pump overnight.
100 ml 0.9% NaCl water solution is added and let stand 4 hours. The flask is mechanically shaken for 30 minutes, and the contents vibrated by ultrasonic 500 W vibration 5 minutes using a Ti tip. These liposomes are used in example 3.
EXAMPLE 2
Peptides and proteins typically contain many amino groups. At least one is terminal, and there might be several lysine side chains, each having one amino group. These can be functionalized with carboxylic acid active ester, such as NHS ester. Alternatively, NHS ester can be created in situ.
1 pmol protein or peptide is dissolved into 10 ml PBS. 10 pmol polyethylene glycol diacid is added (1ml 10 mM solution). Similarly, 10 pmol NHS, and 1 pmol N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) is added (1ml 10 mM solution, and 1ml 1 mM solution, respectively). After 10 min 1ml more 1 mM solution of EDC is added. After 1 hour the recti on mixture is transferred into 100 ml Sephadex G-100 column. The column is eluted with Tris buffer that is pumped with peristaltic pump 5 ml/min. After about 30 min the PEG-peptide has peak value. Fractions (0.5 ml) are collected into microcentrifuge tubes. 10 pl samples are diluted into 1 ml, and absorbance is measured at 260 nm. PEG-peptide is eluted first, then unreacted peptide, and finally unreacted PEG diacid. PEG-peptide fractions are combined and used in Example 3. This reaction is repeated for each protein or peptide separately. These PEGs are carboxylate terminated. Biotin terminated PEG-peptides are obtained by using biotin-PEG carboxylate as a starting material.
The peptide solutions/suspensions can be lyophilized use, such as in Example 3.
Preparation of PEGylated polypeptides
For exemplification, the PEG moiety is 24 units long. Any size PEG can be employed. For example, longer PEG moieties can be employed. If needed, click chemistry can be used to extend the PEG spacer. In an exemplary embodiment, 1 g of t-BOC-lysine (Sigma- Aldrich) is dissolved into acetonitrile/methanol 20: 1. Dicyclohexyl carbodiimide is added. After 1 h, the mixture is filtered to remove dicyclohexyl urea, and the filtrate is concentrated in a rotary evaporator to 10 ml. The product is purified using HPLC. The purified product is depicted in Figure 5C.
117 mg (0.1 mmol) Azido-dPEG24-acid is mixed with 25 mg (0.1 mmol) t- BOC-lysine methyl ester in 10 ml of acetonitrile. 21 mg (0.1 mmol) Dicyclohexyl carbodiimide is added. After 1 h the mixture is filtered, and 1 g of trifluoroacetic acid is added. After 30 min the mixture is purified with HPLC. The purified product can be used in peptide synthesis to produce peptides with one or more Pegylated lysines. For purposes herein, the PEG moieties are for linkage to the liposomes. The purified PEGylated peptide product is depicted in Figure 5B. Generally, the most C-terminal lysine is pegylated. As needed the lysine for pegylation is one that will not eliminate activity.
As an alternative, the peptides can be produced by other methods, such as by recombinant expression in a suitable host, such as E. coli. Lysines for which pegylation could affect or eliminate activity can be replaced with a conservative amino acid replacement, such as Arg, Glu, Gin, His).
EXAMPLE 3
Liposomes from Example 1 and applicable PEG-peptides from Example 2 are combined. 10 pmol NHS, and 1 pmol N-(3-dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (EDC) is added (1ml 10 mM solution, and 1ml 1 mM solution, respectively). pH is adjusted to 9 using sodium carbonate. After 20 min 1ml more 1 mM solution of EDC is added. After 1 hour the rection mixture is transferred into 100 ml Sephadex G-100 column. The column is eluted with Tris buffer that is pumped with peristaltic pump 5 ml/min. After about 20 min the peptide-liposomes have the peak value.
EXAMPLE 4
Liposomes are prepared as in Example 1, except, 5 mg of PE is used 2 mg of biotin-PEG-NHS (Sigma-Aldrich) is added, and pH adjusted to 9 using sodium carbonate. After 1 hour the mixture is dialyzed against PBS. After 6 hours streptavidin in PBS added (same amount as lipid in liposomes). Excess streptavidin is removed by Sephadex G-100 column.
The peptides are functionalized similarly with biotin-PEG-NHS, and also dialyzed. These biotin-PEG-peptides are added into liposome solution, and will bind spontaneously. Unbound peptides can be removed by a Sephadex column.
Similarly, the biotin terminated oligonucleotide can be bound to the streptavidin liposomes. The peptides can be conjugated to biotin terminated oligonucleotides that are complementary to those on the liposomes.
EXAMPLE 5
A. Peptide Fabrication
As detailed in the Examples above, Fig. 1 schematically depicts a short segment of a polypeptide. Amino acids k, 1, and m are lysines, and can be separated from each other by several amino acids. In normal peptide synthesis terminal carboxylic group is protected by a methyl group that is removed by basic hydrolysis before the next step. The methyl ester of next amino acid is added together with a condensing agent that often is dicyclohexyl carbodiimide (DCCI). The polypeptide is often synthesized on a solid phase surface onto which the growing peptide chain is chemically attached. This allows the washing of the unused reagents and soluble reaction products off.
When lysine is added into a polypeptide chain, the epsilon amino group must be protected. Possible protective groups are allyloxycarbonyl and trityl groups. These are orthogonal to other protective groups. In some instances, the Epsilon-amino group of lysine can be reacted with carboxyl terminated PEG. The bonding will be an amide bond that renders the amino group totally inert. The other advantage is that the peptide contains a PEG in a desired site. The other end of PEG should have a functional group that can be used for the conjugation with a liposome. The functional group can be acetylene that can be reacted with azide that is in the liposome (click chemistry), or alternatively azide group, and the liposome has an acetylene. The other lysines can have conventional protective groups, unless several PEGs are intended for the polypeptide. No separate PEGylation step is needed, since the polypeptide as synthesized is PEGylated. The peptide is synthesized in PEGylated form. Fig.2 shows the product, in which lysine 1 is PEGylated, and PEG has an azide group at the end. Azide, as exemplified below, can be used to couple the peptide to a liposome that has acetylene group on the surface (Click chemistry).
If all lysines in a polypeptide happen to be in the active site, PEG-lysines can be added either in amino or carboxylic end of the polypeptide. This kind of addition can be done anyway in order to make the binding of the polypeptide with liposome stronger.
In the Examples above, the PEG is 24 units long. Longer PEG moieties can be used, and/or click chemistry, such as discussed below, can be used to extend the PEG spacer.
B. Liposome Fabrication
Fabrication methods for liposomes are well-known to those of skill in the art. Liposomes can be fabricated in many ways, including heating methods, membrane contractor, and vertic flow focusing (VFF). See, e.g., the following references for each method: (1) Heating method: Mozafari M.R., Reed C.J., and Rostron C., 2002. Development of nontoxic liposomal formulations for gene and drug delivery to the lung. Technology and health care, 10 342- 344; (2) Membrane contractor: Jaafar- Maleej, C., Charcosset, C., and Fessi, H., 2011. A new method for liposome preparation using membrane contractor. Journal of liposome research, 21(3), 213- 220; and (3) Vertical Flow Focusing (VFF): Hood, R.R., and DeVoe, D.L., 2015. High-throughput continuous flow production of nanoscale liposomes by microfluidic vertical flow focusing. Small, 11(43), 5790-5799.
Another advantageous method is high pressure homogenization, which is readily scaled up to industrial production, and produces small (100 nm) liposomes of uniform size and compositions; and high concentrations (up to 15% by weight) can be achieved.
Liposomes that contain a DBCO group can be prepared. The DBCO group reacts with azido-tagged peptides. PEG-phospholipids are commercially available, such as, from Pure Peg (see, biochempeg.com). One end of the PEG is attached to a phospholipid, and the other end of the PEG has a dibenzocyclooctyne (DBCO) group for click chemistry to link to the peptides.
DBCO reagent is a well-known class of click chemistry labeling reagents that react with azide-tagged molecules. DBCO groups can exclusively react with azide- tagged molecules. 2K, 3.4K, and 5K, corresponding to molecular weights of 2.9 kD, 4.3 kD, and 5.9 kD, respectively, of these compounds are available. Equal molar amounts of each is used. For example, if 1 mmole (760 mg) of phosphatidyl choline is used, the total amount of DBCO phospholipid is 0.12 mmole (i.e. 0.04 mmole of each. 0.01 mmole is 29 mg (2K), 43 mg (3.4K), and 59 mg (5K). 0.04 mmoles is 116 mg, 172 mg, and 236 mg, respectively.
In this example, the total weight of all lipid components is 1274 mg. 30 ml of buffer is used to disperse this amount as liposomes. The minimum volume to disperse this amount of liposomes is about 10 ml.
For coupling of peptides, lOx more of each peptide than used in the following example (or other peptides therapeutics, including any described herein) of peptide fabrication can be used. Azidopeptides couple spontaneously with DBCO liposomes.
3 ml of the resulting compositions contains as much of each peptide as occurs in blood. Smaller amounts, however, can be used for administration. Thus, for example, about 1 ml to 3 ml of the liposomes displaying the peptides can be used.
C. Preparation of liposomes displaying peptides
1. 1 g of t-BOC-lysine (Sigma- Aldrich) is dissolved into acetonitrile/methanol 20: 1. Dicyclohexyl carbodiimide is added. After 1 h the mixture is filtered to remove dicyclohexyl urea, and the filtrate is concentrated in rotary evaporator to 10 ml. The product is purified using HPLC.
117 mg (0.1 mmol) Azido-dPEG24-acid is mixed with 25 mg (0.1 mmol) t- BOC-lysine methyl ester in 10 ml of acetonitrile. 21 mg (0.1 mmol) Dicyclohexyl carbodiimide is added. After 1 h the mixture is filtered, and 1 g of trifluoroacetic acid is added. After 30 min the mixture is purified with HPLC. The purified product (Fig. 3) can be used in the peptide synthesis.
2. Cholesterol-PEG-DBCO constructs are readily available from, for example, creativepegworks.com, MW 2k, 3.4k, 5k, 10k, and Ik. These constructs react with azide in copper free click chemistry.
Cholesterol MW is about 400g/mol. Liposomes can contain 40 mol % of cholesterol. 1/40 of this can be PEG-DBC0 derivatives. Out of 100 mg cholesterol, 75 mg actual cholesterol, 30 mg C-PEG-DBCO MW 2k, 48 mg C-PEG-DBCO MW 3.4k, 150 mg C-PEG-DBCO MW 10k. Liposomes should contain 89 % egg PC, and 11 % palmitoyl oleoyl PE of total phospholipids.
3. Egg lecithin MW about 760 g/mol. 1 mmol is 760 mg.
POPE MW about 720 g/mol. Ratio 1 :0.12 is 760 mg Egg PC to 86 mg PE.
0.4 mmol cholesterol is 160 mg. Thus, the resulting liposomes contain 156 mg cholesterol, 2.5 mg MW 2k, 4 mg MW 2k, 6.4 mg MW 3.4k, 21 mg MW 10k of the constructs.
Peptides Amount in the blood Concentration
Peptide 1 = GLP-1 1.68 ug 100 pM
Peptide4= Oxyntomodulin 4.45 ug 200 pM
Peptide5 = Sermorelin 300 ug 30 ug/kg
PeptidelO = Enterostatin/GIP 15 mg 600 nM
There is 10 000 fold difference in the amounts of peptidel and peptide 10.
This will be reflected also in the amounts that are bound to the liposomes. In order to get amounts of different peptides closer to each other the first three are multiplied by 10. The result is:
Peptides Amount in the liposomes Moles
Peptidel = GLP-1 17 ug 5 nmol
Peptide4= Oxyntomodulin 45 ug 10 nmol
Peptide5 = Sermorelin 3 mg 1 umol
PeptidelO = Enterostatin/GIP 15 mg 3 umol
Total amount of phospholipids in the liposomes is 1.12 mmoles in this example. The amount of PEG spacers is 5 pmoles. The amount of peptides can be slightly less than 5 pmoles. See Example above for fabrication of liposomes that can contain 10-fold more peptide.
4. Adding small molecules
Liposomes can carry drugs in many ways. On the outer surface can be chemically bound peptides or other big molecules using spacers, on outer and inner surface molecules can be adsorbed by ionic bonds, bilayer can incorporate lipid soluble drugs, such as steroid hormones, for example testosterone or estrogen, and inner space can contain water soluble drugs. One of these is phentermine hydrochloride that is a known weight loss drug. Using phentermine hydrochloride has an added advantage that it is released slowly after the peptides have been inactivated by proteases. Slow release also suppresses possible side effects that might be observed, if phentermine is digested as a tablet.
To add small molecules, such as phentermine, liposomes and peptides are fabricated separately. In the liposome fabrication buffer, the small molecule such as phentermine hydrochloride, is added at for example 2 mg/ml. Phentermine is not removed from outside of the liposomes after the fabrication. Once fabricated they can be mixed in the quantities indicated above. They couple spontaneously without any catalyst.
The volume of a liposome can be 500 000 nm3 1 mg of water is 1018 nm3. Thus, water in one liposome weighs 5 x 10'13 mg. 1014 liposomes have 50 mg of water, and 1016 liposomes have 5g of water inside. 5g of water is able to dissolve 10 mg of phentermine hydrochloride. If the liposomes are tightly packed, their total volume including water inside and outside would be 10 ml. This would still be reasonable volume for one dose. This is maximally tight packing. A recommended volume for 1016 liposomes is 1000 ml or one liter But the dosage can be lower, and liposomes modified to increase the amount of phentermine.
1016 liposomes have 2 x 10'3 moles or 2 mmoles of phospholipids, i.e., about 1.4 g. If 10 % of total phospholipids are acidic, such as phosphatidic acid or phosphatidyl glycerol, there would be 0.2 mmoles of acidic phospholipids. Each acidic phospholipid molecule can bind one phentermine molecule. Molecular weight of phentermine is 150 g/mol, and 30 mg of phentermine would be bound. Half of that is outside, and would be released fast. The other half will be released slowly. If the radius of the liposome is 100 nm, one bigger liposome would occupy the same place as four smaller (r = 50 nm) liposomes. Amount of the phospholipid would be same, but internal volume would be twice as big as the combined volume of four small liposomes. Thus, to have 10 mg phentermine requires 500 ml of total volume. If the radius is 400 nm, the volume would be 125 ml. But phentermine also is bound by acidic phospholipids, the total amount of phentermine would be 40 mg. By increasing acidic phospholipid concentration to 20 %, phentermine loading can be 70 mg, and total volume could be decreased to 25 ml. The amount used can be substantially lower, since a single dose of phentermine, since a dose of phentermine, when used alone, is about 15 mg to 37.5 mg. In the combinations herein, the dose can be substantially lower. Because phentermine is combined with other drugs, the dosing is lower 10 ml (about 5 mg of phentermine) or significantly less, so that dosage of the resulting liposomes is about 1 ml to 5 ml.
As discussed above, examples of combinations of polypeptides for displaying on or delivering in a delivery vehicle, such as a liposome or an exosome include combinations of at least two or three weight loss drugs and a muscle enhancer, and can further include a small drug, such as an appetite suppressor, an amphetamine family drug, and/or drugs for treating obesity co-morbidities. The small molecules can be fabricated in association with the delivery vehicle, such as the liposomes or exosomes, or can be separately administered. Examples of fabrication of liposomes is detailed in the above examples. Other methods for synthesizing liposomes or other delivery vehicles, such as exosomes, are known and/or apparent to those skill in the art. Exemplary combinations of peptides and exemplary sequences are as follow: Drug 1 : GLPl/GIPl/Oxyntomodulin + ME* (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 4 + SEQ ID Nos: 5, 8, 44 Drug 2: GLPl/GIPl/Amylin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 7 + SEQ ID Nos: 5, 8, 44 Drug 3: GLP1/GIP1 /Glucagon + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 27 + SEQ ID Nos: 5, 8, 44 Drug 4: GLP1/GIP1/CCK+ ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 11 + SEQ ID Nos: 5, 8, 44
Drug 5: GLP1/GIP1/PYY + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 6 + SEQ ID Nos: 5, 8, 44
Drug 6: GLP1/GIP1 /Leptin + ME (Sermorelin or Tesamorelin or IGF1) SEQ ID Nos: 1, 10, 3 + SEQ ID Nos: 5, 8, 44
*ME= A muscle enhancer such as Sermorelin or Tesamorelin or IGF1 or other muscle enhancing peptide or drug that ameliorates the loss of muscle accompanying weight loss by promoting muscle growth
EXAMPLES 6-9
For the following Examples, it is understood that for purposes herein, the peptides are provided on delivery vehicles, such as liposomes and exosomes or other lipid particles. The delivery vehicles, such as liposomes, display at least two peptides for weight loss, and at least one for muscle enhancement for administration. Alternatively, delivery vehicles displaying a single peptide are prepared, and combinations thereof, each displaying one peptide are administered together in separate compositions or in co-formulations. The peptides and regimens can be designed in accord with the Examples below and description herein. As described herein, the compositions can be provided in pens for injection. In some embodiments, the compositions can be formulated for other routes of administration, such as mucosal and, for some vehicles, oral, depending upon the particular delivery vehicle.
EXAMPLE 6
Evaluation of FDA Approved Weight Loss Medications Comparative Example
This Example provides weight loss and other data achieved with existing weight loss drugs/medications and protocols for comparison with the results achieved using the combinatorial method provided herein in which combinations of drugs are administered, and in which the combinations can be rotated.
For comparison with results set forth in Example 6, and described throughout, weight loss in subjects treated with a variety of FDA approved weight loss medications and off label use of other medications that result in weight loss, and bariatric weight loss surgery (z.e., gastric bypass surgery) are described. Results are shown in Table 14, which sets forth, the length of the protocol, the average weight loss in percent of total weight, and the percentage of participants who achieved either greater than 5% and 10% of total weight loss at the end of the study. The results for each medication and gastric bypass surgery are shown. The highest weight loss percentage was achieved after gastric bypass surgery. Treatment with the various FDA approved weight loss medications and off label use of other medications resulted in weight loss, ranging from approximately 6% to 11% loss.
Table 14: Weight reduction comparison among FDA approved weight loss medications
Of these treatments, gastric bypass is the most effective weight loss protocol. It, however, is fraught with adverse side-effects, including, for example, chronic malabsorption, risk of internal hernias, dumping syndrome, and weight rebound. The combinatorial methods provided herein as exemplified in the Examples, provide results comparable to gastric bypass surgery, but without the need for invasive surgery.
EXAMPLE 7
Weight loss and Safety Regimen using a Combination Pharmacological Treatment
This example describes and details exemplary combinatorial weight loss protocols and the results achieved. In general, the protocols are designed mimic the metabolic and physiological effects of bariatric weight loss surgery (z.e., gastric bypass surgery) by selecting combinations medications and treatments that mimic the metabolic and physiological effects of bariatric weight loss surgery (z.e., gastric bypass surgery).
To achieve this a set of weight loss drugs are selected and combinations of the drugs are administered. The combinations of drugs are a subset of the selected drugs. Different sets of combinations of the drugs can be rotated. By virtue of the combinatorial methods herein development of tolerance or desensitization to the medications/ drugs is avoided. As a result, the ceiling of about a 5% to 10% weight loss that occurs with monotherapy protocols, is avoided. Weight loss comparable to or more than weight loss surgery, such as gastric bypass, is achieved. The combinatorial protocol exemplified below and described herein employs combinations of drugs include that peptides that are involved in regulation of the gutbrain axis. As shown herein, these protocols are more effective than the monotherapies.
A weight loss regimen, described below, in which combinations of medications described below, were administered to human subjects. The protocol was conducted in Punta Gorda, Florida. 96 subjects signed informed consent documents and were enrolled in the weight loss regimen. a. Subjects
The protocol was performed with the consent of each participant. Each participant understood that medications provided were being used “off label” and that they could opt out for standard of care, including monotherapy or counseling on diet and exercise. Benefits of the protocol were explained to the participants prior to initiation of the protocol, participants were counseled on general exercise, nutrition, and portion control, and medications were adjusted as needed. All subjects had failed standard of care protocols for weight management, which included intervention (z.e., surgical intervention) in some subjects.
67 female participants ages ranging from 35-78 years of age and 29 male participants ranging from 46-75 years of age finished the initial 3-month period (Table 15). Thus, of the 96 total participants, about 70% were female and 30% were male, and the average age was 59 years old. For comparison, the average age of a bariatric subject is 42 years of age and 83% of subjects are female (see, American Journal of Nursing (2012) 112(9): 26-36; Turchiello da Silva et al., Arq Bras Cir Dig. 28(4): 270-273 (2015)).
Subjects were seen once weekly for the first 4 weeks, and then once every two weeks, for six visits, for a total of 10 visits over 16 weeks. Vital signs and side effects were assessed at each visit. No participants stopped the regimen during the initial 3- month period due to side effects. The most reported side effects include constipation (40% of participants), dry mouth (35% of participants), tremulousness (10%) and insomnia (5%). One male and one female were discontinued prior to completion of the first month due to social circumstances that prevented timely follow-ups. These subjects were excluded from the demographic information, set forth in Table 15, below.
38% of subjects who completed the initial 12 weeks of the regimen withdrew after the initial 12 weeks. Most participants who decided not to progress past the initial 3-month period did so because of limited finances. For example, one couple did very well in the initial 12-week period and decided they no longer needed any further follow ups as they had acquired all the information they needed to maintain their weight loss. The number of subjects who completed the nine-month (36 week) period is set forth in Table 16, below:
Table 16. Percent of Subjects Continuing to the End of the Protocol (at 36 weeks)
Because the participants were approximately 20 to 25 years older than the typical surgical weight loss subject, they had additional medical issues more common to a geriatric population. These issues can hinder weight loss or make it more challenging to lose weight, and include, for example: 1) the fact that specific habits for weight gain have had longer times to set; 2) sarcopenia due to aging that significantly slows down “metabolism;” and 3) orthopedic issues, such as osteoarthritis, which can limit mobility, which is important in weight loss and weight loss maintenance. b. Subjects’ Diet and Exercise
Subjects received exercise and nutrition counseling, adjustment of medications and assessment of vital signs at each visit for the first three months (12 weeks) of the protocol.
1) Diet
Subjects were instructed to adhere to a liberal diet that included low carbohydrate and high protein in the ratio of 20% carbohydrates, 50% protein, and 30% fats, when possible. After 12 weeks, subjects were instructed to increase the proportion of carbohydrates. Subjects were not instructed to count calories; subjects were instructed to be cognizant of portion size/control. Estimated calorie count for the average subject ranged from approximately 1500-2000 kcal/day.
2) Exercise
About 70% of female and 75% of male subjects had some advanced orthopedic limitation that reduced the time and intensity of exercise. About 50% of subjects, due to advance age and musculoskeletal issues, had to find exercise alternatives and may have reduced exercise times. Subjects were instructed to start slowly and minimally complete at least 20 minutes of exercise, three times per week. About 50% of male subjects were supplemented with testosterone. All of the subjects were administered sermorelin to increase release of growth hormone, including in the combinatorial therapy as a muscle sparing/enhancing peptide. c. Assessment of Weight Reduction and Body Changes
Weight and BMI are poor discriminators of health. Body weight, and, thus, weight loss can be composed of water, fat and muscle and at different ratios. The higher proportion of fat relative to muscle indicates an unhealthy state that can promote inflammation and insulin resistance. Conversely, having a higher ratio of muscle mass is associated with lower insulin resistance and improved insulin sensitivity (Srikanthan etal., J Clin Endocrinol Metab. (2011) 96(9):2898-903).
Unlike previous studies and programs, which follow weight loss (z.e., total pounds lost) and body mass index (BMI: kg/m2), which are clinical measurements used to determine if subjects are overweight or obese, the combination therapy protocol in the Example assessed total weight loss, fat loss, and muscle loss/gain, which, in combination, is a more accurate predictor of health status (Gomez- Ambrosi, etal., Obesity 19, 1439-1444 (2011); Gomez -Ambrosi et al, Obesity 19, 1439-1444 (2011); Kim et al., Diabetes Care 34:504-506 (2011)).
Weight, fat and muscle mass were assessed using the InBody 520™ body composition analyzer (research grade). The data presented below are based on analyses conducted with data from the InBody 520™ body composition analyzer (Biospace, Inc.; Los Angeles, CA USA), which assesses shifts of muscle to fat mass and shifts in water weight. The InBody 520™ analyzer assesses weight, lean body mass, body fat mass, body water balance, body mass index (BMI), percentage of body fat, and muscle mass, using bioelectrical impedance. These parameters were assessed during the 36 weeks of the protocol. Subjects were weighed and assessed using InBody 520™ body composition analyzer at weekly intervals for the first four weeks, then once every two weeks for the completion of the initial 3 -month period. d. Combinatorial Pharmacological Regimen
Subjects were administered or self-administered the medications in the protocol by the appropriate route (z.e., subcutaneous injection or oral tablet/capsule) for 36 weeks. Subjects initially were administered a combination of: 1) a GLP-1 agonist (sold under trademarks Trulicity®, B-cise®, Ozempic®, and Victoza®), 2) Phentermine, 3) Liothyronine, 4) topiramate (sold under the trademark Topamax® carbonic anhydrase inhibitor), and 5) Sermorelin. The starting dosages are listed below in Table 17. The dosages were adjusted throughout the treatment period based on the subject response (z.e., weight loss), tolerability, volume of distribution, subject weight, and at the discretion of the treating physician. For example, the dosage of the GLP-1 agonists Trulicity® and Ozempic® was adjusted depending on the response to the medications. The dosage of the Bydureon BCcise® GLP-1 agonist was not adjusted since it is only produced in a 2 mg injectable dose. Phentermine was increased depending on subject response, to, for example, one 37.5 mg tablet in the morning and one-half tablet (18.75 mg total) at 2 p.m. Liothyronine, which had a starting dosage of 25 mcg, was increased to up to 100 mcg. Topamax® carbonic anhydrase inhibitor, which had a starting dosage of 25 mg, was increased to 100 mg as deemed appropriate. If no progression was noted on the initial medications listed above, then the following medications were added to the protocol: Acarbose, Januvia® dipeptidyl peptidase-4 (DPP-4) inhibitor, Invokana® sodium-glucose cotransporter 2 (SGLT2) inhibitor, and Farxiga® (dapagliflozin) in the dosages listed in Table 17, below. For example, if weight loss progress was slow and a subject was consuming large amounts of carbohydrate-rich foods, the alpha-glucosidase inhibitor, Acarbose, was added to the regimen. In another example, if the subject had normal renal function, then a SGLT2 agonist (such as a SGLT2 agonist sold under the trademark Invokana® or Farxiga® or Januvia®) was administered (SGLT2 is a low affinity sodium/glucose transporter). This was added in addition to medications i-iv and ix, below. The regimen provided a minimum of four medications, and more medications were added and/or the dosages were modified as required in subsequent visits.
Table 17: Exemplary medications for regimens i. Exemplary Regimens In some examples, medications i-iv and ix, listed above, were given initially and one or more of the initial medications was discontinued; Topamax® carbonic anhydrase inhibitor was the least tolerated, and was discontinued in some cases. In other examples, the regimen began with administration of medications i-iv and ix and other medications were added; additional medications were administered at week 2 or week 7, for example, if the subject was not responding and needed an extra pharmacological ‘push.’
As noted above, the medication dosages were adjusted throughout the treatment period as needed, and as agreed to between the treating physician and the subject. For example, if a subject was losing at least four pounds per week, typically, no dosage change was made and no new medications were added. For larger subjects (z.e., starting weight over 300 pounds) who were losing less than 6 pounds per week, medication dosages were increased or additional medications were added to the regimen. The hierarchy of decision making included 1) increase the dosage of the medications to the maximum allowable dosage or to the level of tolerability of the medication; and then 2) add a fifth or sixth medication, as needed to increase weight loss. ii. Representative Subjects
The regimens of a representative subset of subjects are set forth below. The exemplary regimens also include examples of modifications made to the combination therapy, including the modifications described above.
For Example, subject 1, a male, received the medications set forth in Table 18, below (dosages are per day):
Table 18: Weight loss medications, phentermine, liothyronine, and a GLP-1 agonist
(sold under the trademark Trulicity®), were increased based on slower initial weight loss than desired. For example, phentermine was increased from i tablet per day to 1 tablet per day after 2 weeks due to less than desired weight loss. Phentermine dosage was further increased to 1 Yi tablets per day Subject 1 had a starting weight of 385.6 pounds and weighed 241.5 pounds at the end of the 36-week weight loss protocol. Weight (pounds), fat and muscle loss of subject 1 after 12 weeks and 36 weeks on the weight loss protocol are set forth in
Table 19 below:
Table 19:
In another example, subject 2, a female, received the medications set forth in
Table 20, below:
Table 20:
Medications, such as phentermine, and liothyronine were increased based on slower than desired initial weight loss. After 8 weeks on phentermine, phentermine was stopped and diethylpropion was rotated in, and subject took 25 mg diethylpropion per day for 4 weeks (weeks 13-16). At week 17, subject ceased taking Diethylpropion and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
Subject 2 previously had received a sleeve gastrectomy procedure for treatment of obesity. Following the procedure, Subject 2 regained weight and participated in the instant protocol to lose additional weight. Subject 2 had a starting weight of 168 pounds and weighed 142.6 pounds at the end of the 36-week weight loss protocol. Weight (pounds), fat and muscle loss of subject 2 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 21 below: Table 21:
In another example, subject 3, a male, received the medications set forth in
Table 22, below:
Table 22:
Weight loss medications (z.e., phentermine, liothyronine) were increased based on slower than desired initial weight loss. After 8 weeks on phentermine, phentermine was stopped and Mirabegron was rotated in, and subject took 25 mg Mirabegron every other day for 4 weeks (weeks 13-16). At week 17, the subject ceased taking Mirabegron and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
Subject 3 had a starting weight of 251.5 pounds and weighed 197.3 pounds at the end of the 36-week month weight loss protocol. Weight (pounds), fat and muscle loss of subject 3 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 23 below:
Table 23:
In another example, subject 4, a male, received the medications set forth in
Table 24, below:
Table 24:
Weight loss medications (ie., phentermine, liothyronine) were increased based on slower than desired initial weight loss. Subject 4 had a starting weight of 489 pounds and weighed 395 pounds at the end of the 36-week month weight loss protocol. Weight, fat, and muscle loss of subject 1 after 12 weeks and 36 weeks on the weight loss protocol are set forth in
Table 25 below: Table 25:
In another example, subject 5, a female, received the medications set forth in
Table 26, below:
Table 26:
Weight loss medications (ie., phentermine, liothyronine) were increased based on slower than desired initial weight loss. After 8 weeks on phentermine, phentermine was stopped and Phendimetrazine was rotated in, and subject took 25 mg Phendimetrazine every day for 4 weeks (weeks 13-16). At week 17, subject ceased taking Phendimetrazine and resumed Phentermine treatment at 1.5 tablets per day, for the rest of the protocol.
Subject 5 had previously received a gastric bypass procedure for treatment of obesity. Following weight loss from the procedure, Subject 5 regained weight and participated in the instant protocol to lose additional weight. Subject 5 had a starting weight of 188.5 pounds and weighed 149.3 pounds at the end of the 36-week weight loss protocol. Weight (pounds), fat and muscle loss of subject 1 after 12 weeks and 36 weeks on the weight loss protocol are set forth in Table 27 below:
Table 27: e. Study Results
Subjects administered the combinatorial therapy were assessed for total weight loss, fat loss, and muscle composition using the InBody 520™ body composition analyzer (Biospace, Inc.; Los Angeles, CA USA). The results are set forth in tables 28 and 29 and are detailed below.
The combined average body weight for males and females at the start of the protocol, before administration of the combinatorial pharmacological therapy, was 122 kg. The results show that the average subject lost 31.5% fat at 36 weeks following the start of treatment. Female subjects lost less total fat and percent fat than male subjects. 36 weeks after the start of treatment, female subjects lost an average of 12.1 kg, and 30.3% fat, and male subjects lost an average of 20.4 kg, and 31.5% fat. Weight loss progressed over time in male and female subjects; total body fat loss continued to decrease from 21.4% and 22% at 12 weeks to 30.3% and 31.5% at 36 weeks for females and males, respectively. Subjects lost approximately 10 to 15 kg in the first 12 weeks; several male subjects lost 7-8 pounds (about 3-4 kg) of body fat in one week. The results show that subjects lost an average of 22% fat mass after 12 weeks of treatment with the pharmaceutical combination administered in accord with the protocol/regimen described herein; this increased to 31.5% fat mass lost after 36 weeks. Given the differences in the demographics (ie., age) of a typical vertical sleeve gastrectomy subject and the subjects, the weight loss induced by the regimen described herein is similar to or greater than the 2-3 pounds (about 0.9-1.3kg) of weight loss per week or approximately 30 to 40 pounds (about 13-23 kg) the first 12 weeks after weight loss surgery. Thus, the instant regimen shows results similar to a vertical sleeve gastrectomy (26.8 kg vs. 13-23 kg) in a population of subjects that are older and, thus, can have more challenges in losing body fat. The instant regimen shows results that are better than mono- or dual therapy for treatment of obesity (see, e.g., Example above).
Total body weight lost, detailed above, includes fat weight, muscle weight and water weight. The studies conducted herein also assessed the amount of fat and muscle loss, using the InBody 520™ body composition analyzer. Analysis of the bioelectrical impedance data shows that the percent body weight lost after treatment with the combinatorial therapy was not due to a concomitant loss in muscle mass. Subjects treated with the combinatorial therapy described herein lost 14% and 19% total body weight at 12 and 36 weeks, respectively. The muscle loss was only 2% and 3% at these time points, whereas the fat loss was 22% and 31.5% at these time points. These results indicate that at 12 and 36 weeks, the fat loss accounted for a greater percent of the total weight lost. Taken together, these results show that the combinatorial therapy for weight loss described herein, showed high overall weight loss and preferentially decreased fat compared to muscle.
Assessment of all of these metrics (e.g., total weight loss, fat loss, and muscle loss), more accurately reflects improvement in health achieved with this combinatorial therapy. Preserving muscle mass is important for improving health, as it aids increased insulin sensitivity and increases the overall well-being of the subject. The results show that females in the instant protocol, who, as a group, had a higher average age from those in previously assessed weight loss trials, showed improved percent muscle mass between 12 and 36 weeks; the muscle loss at 12 weeks was 6% and at 36 weeks was 5%. The results also show that males in the instant protocol, who, as a group, had a higher average age than in previously assessed weight loss trials, showed improved percent muscle mass between 12 and 36 weeks; the muscle loss at 12 weeks was 5% and at 36 weeks was 3%. Most subjects had limited resistance training education but were encouraged to do resistance training at least three times per week.
Older people experience sarcopenia, a progressive skeletal muscle disorder which accelerates muscle mass loss, more commonly due to the lack of anabolic hormones. Notwithstanding this, during this catabolic process, an improvement of 2% (males) and 1% (females) in muscle mass was achieved; no muscle mass was lost between 12 weeks and 36 weeks on the instant protocol despite a high loss of weight overall. Comparable data for muscle mass loss in weight loss surgery subjects is not readily available; most weight loss studies assess only excess weight loss; muscle mass loss or gain after Roux-en-Y or vertical sleeve gastrectomy generally is not reported.
Table 29: Results after Treatment with Combinatorial Therapy compared to baseline
The results show that treatment with a combinatorial therapy protocol, in which combinations of medications that target different pathways are administered, and also one in which the combinations are periodically rotated, such as every two to 4 weeks, results in total weight loss and fat loss that is not achieved with previous pharmaceutical weight loss treatments; the results are comparable to those achieved by a vertical sleeve gastrectomy. The protocol also included assessing fat mass loss/gain and muscle mass loss/gain. The results of 22% fat mass loss in 12 weeks is a substantial amount of weight loss in a geriatric cohort of participants who generally have hormonal and physical barriers to achieving weight loss. EXAMPLE 8 Comparative Example
This example compares the weight loss in subjects in the weight loss regimen described in the Example above using combinatorial pharmacological treatment described herein to other available pharmacological monotherapy treatments.
A. Combinatorial Pharmacological Treatment compared to Semaglutide (sold under the trademark Wegovy®) weight loss medication
Wegovy® semaglutide weight loss medication for subcutaneous injection was approved by the U.S. Food and Drug Administration in June 2021, for chronic weight management in overweight and obese adults who also have at least one weight-related comorbidity (e.g., high blood pressure, type 2 diabetes, or high cholesterol). Semaglutide is a GLP-1 incretin hormone that plays a role in appetite and digestion. Weight loss in subjects administered Wegovy® semaglutide weight loss medication was assessed previously in at least four studies (results detailed below in Table 30) studies 1, 3 and 4 were performed in non-diabetic subjects, and study 2 was performed in diabetic subjects. Study 1 was performed in subjects with obesity or overweight with a comorbidity. Study 2 was performed in subjects with obesity or overweight with type 2 diabetes. Study 3 was performed in subjects with obesity or overweight with a comorbidity who were undergoing intensive lifestyle therapy. Subjects treated with Wegovy® semaglutide weight loss medication for 12 weeks lost an average of 5.7% of their body weight (combined results from studies 1-4).
Table 30, below, is adapted from HIGHLIGHTS OF PRESCRIBING INFORMATION for WEGOVY (semaglutide) injection, for subcutaneous use, Initial U.S. Approval: 2017, revised 06/2021, and sets forth the Wegovy® semaglutide weight loss medication studies 1-3:
Table 30:
LSMean = least squares mean; CI = confidence interval * pO.OOOl (unadjusted 2-sided) for superiority.
The results from the STEP 1 through 5 trials showed that semaglutide is superior at weight reduction when compared with placebo. STEP 2 compared 2.4 mg semaglutide with 1.0 mg semaglutide and determined that 2.4 mg semaglutide cause more significant weight loss than 1.0 mg semaglutide. STEP 4 investigated the discontinuation of semaglutide treatment and found that those who were started on placebo after 20 weeks of treatment with the experimental dose of semaglutide experienced weight gain of around 6 kg. There were no data on actual fat loss or muscle mass loss or retention. These and other results from the studies are set forth in Table
31, below:
Table 31:
Adapted from Singh et al., J Investig Med 70:5-13 (2022) (see also, Wilding et al., N Engl J Med 384:989-1002 (2021); Davies et al., Lancet Diabetes Endocrinol 397:971-84 (2021); Wadden et al., JAMA 325: 1403-13 (2021); Rubino et al., JAMA 325: 1414-25 (2021); Kushner et al., Obesity 28: 1050- 61 (2020)). In contrast, subjects treated with the combinatorial pharmacological therapy described in Example 2 lost 12% of their body weight after 12 weeks, compared to baseline weight. Thus, subjects treated with the combinatorial pharmacological therapy described herein lost more than twice the percent body weight compared to the FDA- approved semaglutide weight loss product. The combinatorial therapy described in Example 2 demonstrates superior weight loss compared to Wegovy® semaglutide weight loss medication treatment after 36 weeks of treatment. Subjects treated with Wegovy® semaglutide weight loss medication for 36 weeks lost an average of 19% of their body weight (combined results from studies 1-4). Subjects treated with the combinatorial therapy described in Example 2 lost 31.5% of their body weight after 36 weeks, compared to baseline weight. The results show that subjects administered the combinatorial pharmacological therapy described in Example 2 exhibited superior weight loss at both time points.
Most studies on obesity, including the studies assessing Wegovy® semaglutide as a weight loss drug, assess total weight loss. Since body fat is the most metabolically harmful tissue type, the instant studies, set forth in Example 2, also assessed body fat loss and muscle mass as a measure of health change; percent fat loss is clinically more relevant to health than total weight loss. As detailed above, the combinatorial pharmacological therapy described in Example 2 was designed to create “leanness” and adjust body composition, which is demonstrated by the preferential loss of fat compared to muscle, and also improved total weight loss compared to an FDA approved weight loss medication, Wegovy® semaglutide weight loss medication.
As demonstrated in Tables 32a, 32b, 32c and 32d, below, the initial weight loss in subjects treated with the combinatorial pharmacological therapy described in Example 2 is superior to the weight loss in subjects treated with the Wegovy® semaglutide weight loss medication; subjects lost more than 7% more weight at 12 and 36 weeks compared to the average subject administered Wegovy® semaglutide weight loss medication.
Table 32a: Treatment with the combinatorial treatment described in the above example result in greater weight loss than semaglutide Differences in the Studies Assessing the Combinatorial Pharmacological Treatment Described in Example 7 and Semaglutide (sold under the trademark Wegovy®) weight loss medication
There were several differences between the weight loss regimen described in Example 2, and previous studies assessing the impact of semaglutide (sold under the trademark Wegovy®) medication on treatment of overweight and obesity. For example, the average age of the subjects treated with the semaglutide (sold under the trademark Wegovy®) medication was 48 years old, compared to 59 years old for subjects administered the combinatorial pharmacological therapy described in Example 2. The starting weight of the subjects administered the combinatorial pharmacological therapy described in Example 2 was higher than the weight of the subjects administered semaglutide (sold under the trademark Wegovy®) medication (122 kg compared to 104.85 kg). The weight loss regimen detailed in Example 2 was conducted in Punta Gorda, Florida, United States, which has an older population, limiting the recruitment population to mostly older subjects. The subjects following the instant protocol were older, heavier and had several exercise-limiting orthopedic conditions. Recruitment for the instant weight loss regimen included several subjects who had failed some form of prior weight loss treatment (i.e., gastric sleeve or Roux-en-Y Gastric Bypass (RYGBP)), and who demonstrated behavioral and musculoskeletal issues that caused their recidivism. Despite the higher average age, which makes it more difficult to lose weight due to decreased mobility and muscle loss; higher starting weight, which can inhibit mobility; and failure to maintain weight loss after previous surgical treatment; subjects administered the combinatorial pharmacological therapy described in Example 2 lost more weight than subjects administered the FDA approved semaglutide (sold under the trademark Wegovy®) weight loss medication.
The dietary restrictions between the Semaglutide (sold under the trademark Wegovy®) weight loss medication trials also were different from the dietary focus in the instant weight loss regimen (see Example 2). In Wegovy® weight loss medication studies 1, 2 and 4, the subjects consumed a diet with a deficit of 500 kcal/day, and subjects in study group 3 consumed 1200-1800 kcal/day followed by 60 weeks of a reduced calorie diet. In the instant weight loss regimen, subjects were not limited to a maximum number of calories, nor instructed to maintain a calorie deficit; subjects focused on portion control rather than calorie count. The exercise instructions between the Semaglutide (sold under the trademark Wegovy®) weight loss medication trials also were different from those set forth in Example 2. In the instant weight loss regimen, few of the subjects could meet the exercise requirement of 100 to 200 minutes per week as set forth in trial 3, or the minimum of 150 minutes per week set forth in trials 1, 2, and 4 of the Semaglutide (sold under the trademark Wegovy®) weight loss medication. The subjects in the weight loss regimen detailed in Example 2, who rarely exercised or had cardiac conditions, were instructed to start on a minimal exercise regimen of 20 minutes, three times per week.
The results from the trials of the Semaglutide (sold under the trademark Wegovy®) weight loss medication show that approximately 6.8% of subjects treated with 2.4mg semaglutide discontinued use due to adverse reactions due to the mediation. Conversely, no participants administered the combinatorial therapy described in Example 2 stopped the regimen during the initial 3 -month period due to side effects.
Despite the potential limitations associated with subject selection, age bias, and a decrease in muscle promoting exercise in the regimen, weight loss after administration of the combinatorial pharmacological therapy described in Example 2 was dramatically better than the FDA approved treatment semaglutide. Additionally, muscle loss was minimal, and low compared to the total body weight loss, during a catabolic process.
B. Comparison of other Weight loss Drugs with the combinations provided herein
The drug tirzepatide is a GLP-l/GIP dual agonist, which activates the GLP-1 receptor, as well as the GIP, which alters energy consumption. Tirzepatide is a once- weekly GIP (glucose-dependent insulinotropic polypeptide) receptor and GLP-1 (glucagon-like peptide 1) receptor agonist. GIP has been shown to decrease food intake and increase energy expenditure. This endogenous hormone known to be upregulated post Rou-en-Y gastric bypass. When combined with GLP-1 greater effectiveness on weight loss was expected.
As discussed above, semaglutide (sold under the trademark Wegovy®) is a once-weekly injection that helps with chronic weight management when used in combination with diet and exercise. The medication was studied in people who did not have type 2 diabetes, but at a higher dose. Semaglutide (under the brand name Wegovy®) is FDA-approved for weight loss in adults with a body mass index (BMI) greater than or equal to 30mg/kg2 alone or 27 mg/kg2 with at least one weight-related comorbidity (e.g., high blood pressure, high cholesterol). Semaglutide has side-effects that render it difficult to tolerate the higher doses required for weight loss. Semaglutide is a GLP-1 incretin hormone that plays a role in appetite and digestion. Incretins — hormones released by your small intestine — are sent out by the body after a meal to help lower your blood sugar by triggering insulin and blocking other sources of sugar. It also slows down how quickly food leaves your stomach (called gastric emptying). These actions result in a feeling of fullness — lowering your appetite leading to weight loss. Medications like GLP-1 agonists are referred to as incretin mimetics since they “mimic” these effects.
This Example compares weight loss results using the combinations herein, as described in the Examples above, with the results of a study of semaglutide and a study of tirzepatide. The efficacy of weight loss for each study and the combinations herein are compared at two different time points, 12 (3mo) weeks and 36 (9mo) weeks. The comparison shows that the instant combinations were more effective for weight loss than either of these drugs.
1. Semaglutide
The semaglutide study was a 68-week trial of 1,961 patients with obesity (BMI >30 kg/m2) or with overweight (BMI 27 kg/m2-29.9 kg/m2) and at least 1 weight-related comorbid condition, such as treated or untreated dyslipidemia or hypertension; patients with type 2 diabetes mellitus were excluded. Patients were randomized in a 2: 1 ratio to either the semaglutide Wegovy® or placebo, both in conjunction with a reduced-calorie diet (-500 kcal/day deficit) and increased physical activity (recommended to a minimum of 150 min/week).
Primary end points:
• Mean percent change in body weight from baseline to week 68’
• Percentage of patients achieving >5% weight loss from baseline to week 68
Secondary End points: • Percentage of patients achieving >10% weight loss from baseline to week 68
• Percentage of patients achieving >15% weight loss from baseline to week 68 • Change in waist circumference from baseline to week 68
• Change in SBP from baseline to week 68
A goal of the study was to evaluate the efficacy of semaglutide solely as a weight loss medication. Participants were recruited based on BMI, excluding those with a diagnosis of type 2 diabetes, except STEP 2, which did not exclude patients with type 2 diabetes. The experimental dose given during the trial was 2.4 mg, delivered subcutaneously once a week. The trial program was completed in March 2021 with a total of five trials; however, the results are still pending for STEP 5.
The results from STEP 1 through 4 showed that semaglutide is superior at weight reduction when compared with placebo. STEP 2 compared 2.4 mg semaglutide with 1.0 mg semaglutide and found 2.4 mg semaglutide to cause more significant weight loss than 1.0 mg semaglutide. STEP 4 investigated the discontinuation of semaglutide treatment and found that those who were started on placebo after 20 weeks of treatment with the experimental dose of semaglutide experienced weight gain of around 6 kg.
Adverse events: Approximately 6.8% of patients treated with the semaglutide at 2.4mg dose discontinued due to adverse reaction to the medication.
Muscle and Fat composition: There were no data on actual fat loss or muscle mass loss or retention.
B. Tirzepatide
Tirzepatide (5 mg, 10 mg, 15 mg) achieved superior weight loss compared to placebo at 72 weeks of treatment in topline results from Eli Lilly and
Company's (NYSE: LLY) SURMOUNT-1 clinical trial, with participants losing up to 22.5% (52 lb. or 24 kg) of their body weight. This study enrolled 2,539 participants and was the first phase 3 global registration trial evaluating the efficacy and safety of tirzepatide in adults with obesity, or overweight with at least one comorbidity, who do not have diabetes. Tirzepatide met the co-primary endpoints of superior mean percent change in body weight from baseline and greater percentage of participants achieving body weight reductions of at least 5% compared to placebo for both. The study also achieved all key secondary endpoints at 72 weeks.
Primary end points:
• Mean percent change in body weight from baseline to week 72
• Percentage of patients achieving >5% weight loss from baseline to week 72
Secondary End points:
• Percentage of patients achieving >10% weight loss from baseline to week 72 Percentage of patients achieving >20% weight loss from baseline to week 72
Table 32c DATA
For the efficacy estimate, participants taking tirzepatide achieved average weight reductions of 16.0% (35 lb. or 16 kg on 5 mg), 21.4% (49 lb. or 22 kg on 10 mg) and 22.5% (52 lb. or 24 kg on 15 mg), compared to placebo (2.4%, 5 lb. or 2 kg). Additionally, 89% (5 mg) and 96% (10 mg and 15 mg) of people taking tirzepatide achieved at least 5% body weight reductions compared to 28% of those taking placebo.
Adverse events: Treatment discontinuation rates due to adverse events were 4.3% (5 mg), 7.1% (10 mg), 6.2% (15 mg) and 2.6% (placebo). The overall treatment discontinuation rates were 14.3% (5 mg), 16.4% (10 mg), 15.1% (15 mg) and 26.4% (placebo).
Muscle and Fat composition: There were no data on actual fat loss or muscle mass loss or retention.
3. Combinatorial treatment
Average age: participant average age was 59 years old, over 70% of females and 75% of males had some advanced orthopedic limitation which would reduce time and intensity of exercise.
Trial design is described in the Example above: As noted, all patients had failed standard of care protocols for weight management to include surgical intervention in some. Patients were instructed on a liberal diet that included low carbohydrate and high protein diet in the ratio of 20% carbs 50% protein and 30% fats when possible. After 3 months they were instructed to increase the carbs. There were no counting calories only portion control. Estimated calorie count for the average patient would range from 1500-2000 kcal/day. As for exercise, over 50% of patients mentioned above due to advance age and musculoskeletal issues had to find alternatives to exercise and may have reduced exercise times. Patients at the minimum were instructed to start slow with at least 20 mins 3xs a week.
Primary end points:
• Mean percent change in body weight from baseline to week 12, and from baseline to week 36.
• Percentage of patients achieving >5% weight loss from baseline to week 12, and from baseline to week 36.
Secondary End points:
• Percentage of patients achieving >10% weight loss from baseline to week 12, and from baseline to week 36.
• Percentage of patients achieving >15% weight loss from baseline to week 12, and from baseline to week 36.
Table 32d DATA
Muscle enhancement: More than 50% of male subjects, were supplemented with testosterone and all males and females had sermorelin added to their regimens as a muscle sparing/enhancing peptide.
4. Differences between the semaglutide study the protocol for the combinatorial study a. The age between the two groups is significantly different. The group in the StarRock Cocktail protocol were on the average 10 years older, heavier and with several exercise limiting orthopedic conditions. The protocol was conducted in Punta Gorda FL, which limited the recruitment population to older patients. The recruitment also included patients who had failed some form, whether gastric sleeve or RYGBP, which posed unique issues, to include behavioral and musculoskeletal issues that caused their recidivism. b. There were few patients that could meet the exercise requirement of 100- 200 min/week in our study group. As noted above, patients who rarely exercised or had cardiac conditions were instructed to start on a minimal exercise regimen of 20 min 3 times a week. c. The dietary restrictions are notably different as well. In study groups 1, 2, and 4 there was a deficit of 500kcal/day and study group 3 of 1200-1800. The focus was on portion control rather than calorie count since large discrepancies in actual calorie count could cause for erroneous data interpretation. d. A focus was on enhancing muscle development through peptide/hormone supplementation, which in effect this reduces percent weight loss as increased muscle mass contributes to total bodyweight.
For this reason, the percent fat loss, which is clinically more relevant to health, is considered the more relevant parameter.
5. Results
As seen in table above, the initial weight loss in comparison to semaglutide is superior in the protocol by more than 7% at 3 months. There was on average a 22% bodyfat drop from baseline with the combinatorial protocol. This became more relevant at 9 months, where even though weight loss is a catabolic process, there was not loss of muscle mass between 3 months and 9 months, and a higher portion of the percent weight loss was fat loss. The percent fat loss increased from 22% to 31.5% during this time.
Even with the limitations above with the patient selection, age bias, and a muscle promoting component, the percentage of weight loss with the combinatorial therapy provide herein was significantly more effective and resulted in minimal muscle loss during a catabolic process.
D. The combinatorial pharmacological treatment compared to other previously characterized treatments Subjects administered the combinatorial pharmacological therapy described in Example 2 show greater weight loss than subjects administered phentermine, orlistat, Qsymia, Locasarin, naltrexone-bupropion, or liraglutide. The results, which are set forth in Table 33 below, compare the total weight loss and percent of subjects who lost greater than 5% (>%5) or greater than 10% (>%10). The last row labeled “Combinatorial Therapy” is the weight loss in subjects administered the therapy detailed herein in Example 2 (N=96). These results show that a combination of medications has results that are comparable to a surgical gastric bypass, but are noninvasive, and that surpass the weight loss associated with monotherapy with known weight loss medications.
Table 33: Treatment with the combinatorial pharmacological treatment described herein results in greater weight loss than previously characterized treatments
EXAMPLE 9
Weight loss and Safety Demonstration using a Rotational Combinatorial Pharmacological Treatment
The rotational combinatorial pharmacological treatment includes a combination of therapeutic and endogenous peptides administered to human subjects. As described in the detailed description, a rotational combinatorial pharmacologic protocol for obesity that mimics the plurality of effects of gastric bypass was designed. The protocol targets multiple orexigenic pathways for downregulation by administering peptide combinations. a. Subjects
A protocol is performed with subject consent and potential benefits and adverse effects of the protocol are identified prior to initiation. Subjects include those that had failed standard of care protocols for weight management and/or that are overweight or obese. b. Assessment of Weight Reduction and Body Changes
Weight, fat and/or muscle mass are assessed before, during and after treatment. For example, weight, fat, and muscle mass are assessed using the InBody 520™ body composition analyzer (research grade), during the treatment period at predetermined intervals, and post-treatment. c. Combinatorial Rotational Therapy
The combinatorial rotational therapy is self-administered by subcutaneous injection or taken orally or by another appropriate route for the particular peptide. Subjects are administered GLP-1 (a 31aa biochemical variant of semaglutide), adiponectin (244aa), Leptin (167aa), Oxyntomodulin (36aa), Sermorelin (29 aa), PYY (34aa), Amylin (37aa), tesamorelin (44aa), Pancreatic peptide e (36aa), Enterostatin/GIP (Gastroinhibitory Polypeptide) (50aa), CCK Peptide (4aa), Vasoactive Intestinal Peptide (28 aa), and/or Glicentin (69aa) in various combinations at the times and intervals set forth in Table 34, below:
Table 34: Exemplary Regimen for Peptide Administration
The peptides are administered in amounts necessary to achieve the following target peptide serum levels: semaglutide 3-250nmol/ml; adiponectin 3-7ug/ml; leptin 6-20ng/ml; oxyntomodulin 12-30pmol; sermorelin (inject 200mcg daily bid); PYY 4- 370pg/ml; Amylin 20-200ng/ml; tesamorelin; pancreatic polypeptide 200-350 pg/ml; enterostatin/GIP 250pg/ml-370 pg/ml; cck 6-50pg/ml; VIP <70pg/ml; and glicentin 8- 40pmol/L. The half-life of the peptides varies, for example, the variant of semaglutide has a half-life of 7 days, adiponectin has a half-life of 2.5 hours, and VIP has a half-life of 2 minutes. Each of the peptides have effects for weight loss and sustaining muscle, and most have anti-glycemic effects to ameliorate symptoms of type II diabetes. e. Results
Subjects administered the combinatorial rotational therapy in accord with the timing set forth in Table 34, above, are assessed for total weight loss, fat loss, and/or muscle composition, for example, by using the InBody 520™ body composition analyzer (Biospace, Inc.; Los Angeles, CA USA).
EXAMPLE 10
Weight loss using a Rotational Combinatorial Pharmacological Treatment
Exemplary rotational combinatorial treatments are described above and detailed in Tables 6.1, 6.2, 6.3, and 6.4. As another example, a subject is treated with a rotational combinatorial weight loss protocol in which a combination of at least two, typically at least three, of drugs, such as those set forth in Table 34, above, are administered for a predetermined time as a first round, and then a different combination, which contains two or three drugs, which can include a drug from the first round, but is a different combination is administered for a predetermined time, followed by a third round, and so on. One or more of the rounds can include a single drug, as long as another round includes at least two, generally at least 3 drugs. The rounds can be repeated after one regimen is completed. Table 35 below provides an exemplary rotational combinatorial regimen for weight loss, and exemplary doses. It is understood that doses can be adjusted for a particular subject depending upon parameters understood by skilled practitioners, such as height, weight, age, sideeffects experienced, and other parameters.
Table 35: Exemplary subject treatment regimen
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Since modifications will be apparent to those of skill in the art, it is intended that invention(s) herein only is/are limited by the scope of the appended claims.

Claims

What is Claimed:
1. A delivery vehicle, comprising a combination of therapeutic peptides, wherein: the peptides are linked to the surface directly or indirectly via a linker or are part of the surface of the delivery vehicle or are in the delivery vehicle; the surface of the delivery vehicle optionally is modified for linkage of the peptides; the combination of peptides comprises at least three different peptides; at least two of the peptides target different pathways and/or have different activities; and the peptides target pathways involved in obesity and/or diabetes, or have activity for treating obesity and/or diabetes or other obesity comorbidity.
2. The delivery vehicle of claim 1, further comprising a small molecule drug for treatment of obesity or an associated comorbidity.
3. A composition, comprising a mixture of delivery vehicles, wherein: each delivery displays at least one therapeutic peptide on the surface and/or contains the at least one peptide; and the composition comprises delivery vehicles that are selected so that the composition comprises at least three different peptides.
4. The composition of claim 3 that is formulated for injection.
5. The delivery vehicle or composition of any of claims 1-4, wherein the delivery vehicle selected from among a liposome, lipid nanoparticle, exosome, and extracellular vesicle.
6. The delivery vehicle or composition of any of claims 1-5, wherein the peptides comprise fat loss and muscle enhancement peptides, wherein muscle enhancement polypeptides reduce or eliminate loss of muscle mass or increase muscle mass.
7. The delivery vehicle or composition of any of claims 1-6, wherein two of the peptides are fat loss peptides, and one is a muscle enhancement polypeptide.
8. The delivery vehicle or composition of any of claims 1-7, wherein the polypeptides for fat loss (PeptideFL) are selected from among:
PeptideFLl=GLP-l, PeptideFL 2=Adiponectin,
PeptideFL3=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide),
PeptideFL9= Glicentin,
PeptideFL 10 = Glucagon,
PeptideFL 11=GRPP,
PeptideFL 12=HGH 176-191,
Peptide FL13= CCK,
PeptideFL 14= Neurotensin,
PeptideFL15= Secretin,
PeptideFL 16= IP1, and
PeptideFL17= MPGF (major proglucagon fragment).
9. The delivery vehicle or composition of any of claims 1-8, wherein the peptides for muscle enhancement (PeptideME) are selected from among:
PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and
PeptideME2=IGFl or human growth hormone to induce IGF1.
10. The delivery vehicle or composition of any of claims 1-9, wherein: a) the polypeptides for fat loss are selected from among:
PeptideFL 1=GLP-1,
PeptideFL 2=Adiponectin,
PeptideFL3=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide),
PeptideFL9= Glicentin, PeptideFLIO = Glucagon,
PeptideFLl 1=GRPP,
PeptideFLl 2=HGH 176-191,
Peptide FL13= CCK,
PeptideFLl 4= Neurotensin,
PeptideFLl 5= Secretin,
PeptideFLl 6= IP1, and
PeptideFLl 7= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement are selected from among:
PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and
PeptideME2=IGFl or human growth hormone to induce IGF1.
11. The delivery vehicle or composition of any of claims 1-10, wherein the peptides comprise GLP-1, Oxyntomodulin, enterostatin/GIP (Gastroinhibitory Peptide); and Sermorelin.
12. The delivery vehicle or composition of any of claims 1-11, wherein the delivery vehicle is a liposome.
13. The delivery vehicle or composition of claim 12, wherein the liposome is a large multilamellar vesicle (LMV).
14. The delivery vehicle or composition of any of claims 1-13, wherein: the delivery vehicle comprises a liposome; and the liposome comprises phospholipids.
15. The delivery vehicle or composition of claim 14, wherein the phospholipids are selected from one or more of phosphatidyl choline (PC), phosphatidyl ethanol amine (PE), and phosphatidyl serine (PS), and phosphatidic acid (PA).
16. The delivery vehicle or composition of any of claims 1-15, wherein: the delivery vehicle is a liposome; and the phospholipid is from a natural source.
17. The delivery vehicle or composition of any of claims 1-16, wherein: the delivery vehicle comprises a liposome; the liposome comprises cholesterol; and the amount of cholesterol is sufficient to increase the permeability of the liposome compared to the liposome that does not contain the cholesterol.
18. The delivery vehicle or composition of any of claims 1-17, wherein: the delivery vehicle is a liposome; the liposome comprises cholesterol; and the molar percentage of cholesterol in the liposome is less than 60%, 50%, 40%, 30%, 20%, 10%, or less.
19. The delivery vehicle or composition of any of claims 1-18, wherein: the delivery vehicle is a liposome; and the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group.
20. The delivery vehicle or composition of any of claims 1-19, wherein: the delivery vehicle is a liposome; the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group; and the reactive group for the coupling reaction is selected from among amino, thiol, maleimide, bromo- or iodoacetyl, pyridyl di thio, carboxylic, hydrazide, p- nitrophenyl carbonate, azide, and alkyne.
21. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; the liposome comprises lipids modified with a reactive group for coupling with a peptide or peptide modified with a reactive group; the reactive group is an amino group that forms an amide bond with an activated carboxylic ester, or is a thiol group that binds with maleimide, bromo- or iodo acetyl, pyridyldithio groups, or is a hydrazide that bind with carbonyl groups, or is p-nitrophenyl carbonate that reacts with amines forming an amide bond, or comprise azide and alkyne group that bind with each other in the presence of a copper ion catalyst, to attach the protein to the liposome.
22. The delivery vehicle or composition of claim 21, wherein the peptides are linked to the liposomes via the bonds formed by reaction of the reactive groups.
23. The delivery vehicle or composition of any of claims 1-22, wherein: the delivery vehicle is a liposome; and the peptide and liposome are linked via an amide/peptide bond or linker, a thioester bond or linker, a disulfide bond or linker, a hydrazone bond or linker, a carbamate bond or linker, and a 1,2, 3 -triazole linker.
24. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; wherein the peptide and/or liposome is/are PEGylated for linking the peptide to the liposome.
25. The delivery vehicle or composition of any of claims 1-20, wherein: the delivery vehicle is a liposome; and the liposome and/or peptide is PEGylated for linkage of the peptide to the liposome.
26. The delivery vehicle or composition of any of claims 1-25, wherein: the delivery vehicle is a liposome; the linkage of the peptide to the liposome comprises a spacer.
27. The delivery vehicle or composition of claim 26, wherein the spacer comprises polyethylene glycol (PEG) and/or and an oligonucleotides bound to the liposome and to the peptide linked to a complementary oligonucleotide.
28. The delivery vehicle or composition of any of claims 1-26, wherein: the delivery vehicle is a liposome; the liposome comprises streptavidin bound to biotin-linked peptide or the streptavidin is bound to the liposome and to biotin-linked peptide.
29. The delivery vehicle or composition of claim 28, wherein: the liposomes are coated with a monolayer of streptavidin and linked to peptides functionalized with biotin-PEG-NHS to form liposomes that display the peptides upon mixing these peptide derivatives with streptavidin liposomes.
30. The delivery vehicle or composition claim 28, wherein: the linkage of the peptide to the liposome comprises a PEG spacer; one end of the spacer is attached biotin, and the other comprises a reactive group, such as an NHS active ester, that easily forms an amide bond with the PEG.
31. A combination of the delivery vehicle or composition of any of claims 1-30 that comprises one or more small molecule drug(s), wherein: the small molecule drug enhances weight loss or treats a comorbidity associated with obesity; and the small molecule is formulated in or with the delivery vehicle or is for administrations separately from the delivery vehicle.
32. The combination of claim 31, wherein the small molecule drug is/are selected from among one or more of: Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate.
33. The combination of any of claims 30-32, wherein the delivery vehicle and small molecule drugs are for administration together in a single composition, sequentially, or intermittently.
34. A container, comprising the delivery vehicle or composition of any of claims 1-30.
35. The container of claim 34 that comprises a pen or a syringe for administering the delivery vehicle.
36. The container of claim 34 or claim 35 that is an autoinjector.
37. The container of any of claims 34-36 that is a multicompartment container, wherein one compartment contains the delivery vehicles or a mixture of different delivery vehicles, which is/are lyophilized; and the other contains a pharmaceutically acceptable vehicle for mixing with the delivery vehicles to produce a composition for injection.
38. A pharmaceutical composition, comprising the composition or delivery vehicle of any of claims 1-30.
39. The delivery vehicle, composition, container, or pharmaceutical composition of any of claims 1-38 for use for treating obesity.
40. A method of treatment of obesity or diabetes, comprising administering a delivery vehicle or composition of any of claims 1-30 and 38.
41. A method of treating a disease, disorder, or condition, comprising administering and rotating combinations of drugs, wherein: drugs include peptides and small molecule drugs; the combinations of drugs comprise delivery vehicles or compositions of any of claims 1-30; the disease, disorder, or condition is obesity and/or diabetes; at least two combinations are rotated for each cycle of treatment; and treatment comprises at least two cycles.
42. A regimen for treating a disease, disorder, or condition, comprising combinations of peptides for use in a rotational combinatorial regimen, wherein: the disease, disorder, or condition is obesity or diabetes, a chronic disease, disorder, or condition that requires treatment for at least 6 months; each combination comprises at least two different peptides that target different pathways or intervention targets; and the combinations of peptides comprise a delivery vehicle or composition of any of claims 1-33.
43. The regimen of claim 42, further comprising a small molecule drug for weight loss and/or for treating a comorbidity associated with obesity.
44. The regimen of claim 43, wherein the small molecule drug is selected from one or more of Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
45. The regimen of claim 44, wherein the small molecule drug is incorporated in or on the delivery vehicle and/or the small molecule drug is coadministration, simultaneously, sequentially, or intermittently with the delivery vehicle(s).
46. The method of claim 41 or regimen of any of claims 42-45, comprising a plurality of cycles of administration of different combinations of peptides and optionally the small molecules, wherein: each combination is administered at least once a cycle; a cycle comprises administration of each combination at least once; a cycle comprises at least two different combinations; a cycle can be repeated a plurality of times; and at least one of the combinations comprises at least two different drugs that target different targets or pathways.
47. The method or regimen of any of claims 41-46, wherein each cycle comprises one delivery vehicle that comprises at least 3 different peptides.
48. The method or regimen of any of claims 41-46, wherein each cycle comprises a mixture of delivery vehicles; each delivery vehicle comprises at least peptide, whereby at least three different peptides are administered in each cycle; and the different delivery vehicles are administered together or serially.
49. The method or regimen of any of claims 41-48, wherein the different vehicles are administered within 24 hours of each other.
50. A method for treating obesity, comprising administering a delivery vehicle of any of claims 1-30, wherein each peptide targets a different pathway or different target for intervention for treatment of obesity.
51. The method or regimen of any of claims 41-50, wherein the combination of peptides mimic effects of gastric bypass.
52. The method or regimen of any of claims 41-51, wherein the combination of peptides on the delivery vehicle comprises at least three selected from among: a peptide that inhibits gastric emptying selected from among one or more of GLP1, Amylin, and Pancreatic Polypeptide Therapeutic; a peptide drug that enhances satiety comprising one or more drugs selected from among glucagon-like peptide- 1 (GLP-1), peptide YY (PYY), amylin, enterostatin/gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glicentin; a peptide that increases insulin release and/or sensitivity comprising one or both of GLP1 and adiponectin; and a peptide that modulates energy expenditure comprising leptin, oxyntomodulin, and glicentin.
53. The delivery vehicle, composition, method, or regimen of any of claims 1-51, wherein the vehicle comprises or displays combinations of peptides selected from among combinations of drugs for fat loss + a drug for muscle enhancement: a) GLPl/GIPl/Oxyntomodulin + Sermorelin or Tesamorelin or IGF1; b) GLPl/GIPl/Amylin + Sermorelin or Tesamorelin or IGF1; c) GLP1/GIP1 /Glucagon + Sermorelin or Tesamorelin or IGF1; d) GLP1/GIP1/CCK+ Sermorelin or Tesamorelin or IGF1; e) GLP1/GIP1/PYY + Sermorelin or Tesamorelin or IGF1; and f) GLP1/GIP1 /Leptin + Sermorelin or Tesamorelin or IGF1.
54. The delivery vehicle, composition, method, or regimen of claim 53, wherein the amino acid sequence of each of the peptides for each of a)-f) are as set forth set forth in the following SEQ IDs, or are variants thereof that have at least 90% or at least 95% sequence identity thereto and retain activity for effecting fat loss or muscle enhancement: a) SEQ ID NOs: 1, 10, 4 or 47 + SEQ ID NOs: 5, 8, 44; b) SEQ ID NOs: 1, 10, 7 + SEQ ID NOs: 5, 8, 44; c) SEQ ID NOs: 1, 10, 27 + SEQ ID NOs: 5, 8, 44; d) SEQ ID NOs: 1, 10, 11 + SEQ ID NOs: 5, 8, 44; e) SEQ ID NOs: 1, 10, 6 or 37 + SEQ ID NOs: 5, 8, 44; and f) SEQ ID NOs: 1, 10, 3 + SEQ ID NOs: 5, 8, 44.
55. The delivery vehicle, composition, method, or regimen of any of claims 1-52 that includes a delivery vehicle that comprises a peptide that results in muscle enhancement and/or a drug or polypeptide that inhibits the myostatin pathway.
56. The delivery vehicle, composition, method, or regimen, wherein the delivery vehicle comprises one or more of sermorelin, tesamorelin and/or growth hormone, and testosterone.
57. The method or regimen of any of claims 41-56, further comprising administering a delivery vehicle that comprises a peptide that promotes intestinal smooth muscle relaxation, such as vasoactive intestinal peptide (VIP).
58. The method or regimen of any of claims 41-57 wherein: the peptides are selected from among:
GLP-1, Adiponectin, leptin, oxyntomodulin, peptide tyrosine-tyrosine (PYY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), vasoactive intestinal peptide (VIP), glicentin, human growth hormone or an active portion thereof or an analog of human growth hormone or an active portion thereof, ephedrine, caffeine, aspirin (EC A), oxyntomodulin, neuropeptide Y (NPY), antimicrobial peptide 2 (LEAP2), vaccine CYT009-GhrQb, the peptide-binding compound Nox-Bl 1, and the ghrelin analog AZP-531 (SEQ ID NO: 15); and/or the peptides are a GLP-1 agonist, an appetite suppressant, a thyroid hormone, a carbonic anhydrase inhibitor, an alpha-glucosidase inhibitor, a dipeptidyl peptidase- R (DPP-4) inhibitor, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a muscle enhancer, drugs that modulate energy expenditure, a GLP-1 agonist, peptides that increase gastric inhibitory polypeptide (GIP), drugs that modulate GIP2, and mitochondrial uncouplers; and the peptides are combined by displaying a plurality on each delivery vehicle or by mixing delivery vehicles that display different peptides.
59. The method or regimen of any of claims 41-58, wherein the peptides and drugs are selected from among: dulaglutide, bydureon, semaglutide, exenatide, liraglutide, phentermine, liothyronine, topiramate (carbonic anhydrase inhibitor), acarbose (alpha-glucosidase inhibitor), sitagliptin (dipeptidyl peptidase-4 (DPP -4) inhibitor), canagliflozin (sodium-glucose co-transporter 2 (SGLT2) inhibitor), dapagliflozin ( SGLT2 inhibitor), sermorelin, mirabegron (beta-3 adrenergic agonist), and amylin.
60. The method or regimen of any of claims 41-59, wherein the peptides and drugs are selected from among: a GLP-1 agonist, phentermine, thyroid hormone, carbonic anhydrase inhibitor, carbonic anhydrase inhibitor, alpha-glucosidase inhibitor, DPP-4 inhibitor, SGL2 inhibitor, muscle enhancer, and an appetite suppressant.
61. The delivery vehicle, composition, method, use, or regimen of any of claims 1-58, further comprising a mitochondrial uncoupler, wherein the mitochondrial uncoupler is provided linked to a delivery vehicle or mixed in the composition or administered as a free molecule not bound to a delivery vehicle.
62. The delivery vehicle, composition, method, use, or regimen of claim 59, wherein the mitochondrial uncoupler is selected from among uncoupling protein 1 (UCP1), a catecholamine, and a small molecule uncoupler, such as 2,4-dinitrophenol (DNP) and BAM15 (N5,N6-bis(2-Fluorophenyl)[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine).
63. The delivery vehicle or composition of any of claims 1-33 for use for treating obesity and/or diabetes.
64. A method of preparing a PEGylated peptide, comprising preparing a PEGylated lysine and adding it to peptide during solid phase synthesis.
65. The method of claim 64, wherein the PEGylated lysine is prepared by reacting the epsilon-amino group of the lysine with a carboxyl terminated PEG moiety via an amide group to produce a PEGylated lysine.
66. The method of claim 65, further comprising adding the PEGylated lysine to a peptide during solid phase synthesis of the peptide.
67. The method of any of claims 64-66, wherein the PEG moiety comprises a functional group for conjugation with a reactive group, such as one on a liposome.
68. The method of claim 67, wherein the functional group is acetylene that is reacted with azide in the liposome (click chemistry).
69. A method of conjugating a polypeptide to a liposome, comprising adding a lysine to a polypeptide during solid phase synthesis by reacting the epsilon-amino group of lysine with a carboxyl terminated PEG moiety via an amide group to produce a peptide comprising a lysine comprising the PEG moiety, which comprises a functional group for conjugation with the liposome; and conjugating the PEGylated peptide to the liposome.
70. The method of claim 69, wherein the functional group is acetylene that is reacted with azide in the liposome (click chemistry).
71. The method of any of claims 64-70, wherein the peptides comprise a is a GLP-1 agonist and a muscle enhancer peptide.
72. The method of claim 71, wherein the peptide is selected from among:
GLP-1 pathway agonist, adiponectin, leptin, oxyntomodulin, PYY (peptide YY), amylin, pancreatic peptide, enterostatin/gastric inhibitory polypeptide (GIP), glicentin, glucagon, GRPP (glicentin-related pancreatic polypeptide), HGH (human growth hormone), CCK (cholecystokinin), neurotensin, secretin, IIP (myo-inositol 1- phosphate), and MPGF (major proglucagon fragment); and/or the peptides are sermorelin, tesamorelin, and IGF1 (or HGH).
73. The method of claim 69, comprising preparing at least three pegylated polypeptides and linking the peptides to one liposome or each to a different liposome, or two to one liposome and the third on a separate liposome.
74. The method of any of claims 64-73, wherein the peptides linked to the liposome or to different liposomes comprise a polypeptide for fat loss, and a polypeptide for muscle enhancement or a polypeptide that inhibits the myostatin pathway.
75. The method of claim 74, wherein: a) the polypeptides for fat loss (FL) are selected from among:
PeptideFL 1=GLP-1,
PeptideFL 2=Adiponectin,
PeptideFL3=Leptin,
PeptideFL4= Oxyntomodulin,
PeptideFL5=PYY,
PeptideFL6= Amylin,
PeptideFL7=Pancreatic peptide,
PeptideFL8=Enterostatin/GIP (Gastroinhibitory Polypeptide),
PeptideFL9= Glicentin,
PeptideFL 10 = Glucagon,
PeptideFL 11=GRPP,
PeptideFL 12=HGH 176-191,
Peptide FL13= CCK,
PeptideFL 14= Neurotensin,
PeptideFL15= Secretin,
PeptideFL 16= IP1, and
PeptideFL 17= MPGF (major proglucagon fragment); and b) the peptides for muscle enhancement (ME) are selected from among:
PeptideMEl=Sermorelin;
PeptideME2=Tesamorelin; and
PeptideME2=IGFl (or human growth hormone to induce IGF1).
76. A liposome, comprising one or more of a fat loss peptide and a muscle enhancement peptide, wherein the liposome is produced by a method of any of claims 69-75.
77. The delivery vehicle, composition, use, method, regimen, or liposome of any of claims 1-64 and 76, further comprising a small molecule drug, wherein the drug is for weight loss and/or a co-morbidity associated.
78. The delivery vehicle, liposome, use, method, or liposome of claim 77, wherein the co-morbidity is metabolic syndrome.
79. The delivery vehicle, composition, use, method, regimen, or liposome of claim 77 or claim 78, wherein the co-morbidity is diabetes, hypertension, dyslipidemia, and heart disease.
80. The delivery vehicle, composition, method, regimen, or liposome, wherein the small molecule drug is Phentermine, Topiramate, Metformin, Empagliflozin, Dapagliflozin, Bexagliflozin, Ertugliflozin, Linagliptin, Canagliflozin, NS-2330, Liothyronine, Diethylpropion, Zonisamide, Albuterol, Clenbuterol, Levothyroxine, Naltrexone, Orlistat, Testosterone Cypionate, and Testosterone Enanthate, and a statin.
81. The delivery vehicle, composition, use, method, regimen, or liposome of any of claims 1-64, and 76-80, wherein the delivery vehicle comprises phentermine or wherein phentermine is combined with a composition comprising the delivery vehicle or liposome for combination therapy.
82. The delivery vehicle, composition, use method, regimen, or liposome of any of claims 1-81, wherein the delivery vehicle is an extracellular vesicle or an LPN, or an exosome, or a liposome.
PCT/US2025/026532 2024-04-25 2025-04-25 Delivery vehicles comprising proglucagon derived polypeptides and anabolic polypeptides and uses thereof Pending WO2025227129A2 (en)

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